Polypeptide of protein P140 and DNAs encoding it

ABSTRACT

The present invention is related to a novel protein p140 polypeptide which is a key protein involved in the signal transmission system of insulin; method for preparation of it; DNA encoding the polypeptide; vector derived with the DNA; host cells transformed with the vector; antibody of the polypeptide; pharmaceutical composition containing the peptide or antibody; method for the prevention and/or treatment of diabetes, which is characterized by tyrosine phosphorylation of the protein p140; agent for the prevention and/or treatment of diabetes, which is characterized by containing a compound which can tyrosine phosphorylate protein p140, as active ingredient and the screening methods of the prevention and/or treatment agent. Tyrosine phosphorylation of protein p140 is an essential step in the induction of hypoglycemia by glucose uptake. Method and agent of prevention and/or treatment based on tyrosine phosphorylation of protein p140 in the present invention not only improve the diabetes-derived hyperglycemic conditions but are also useful for the treatment and/or prevention of diabetes, especially non-insulin dependent diabetes mellitus (NIDDM).

This is a divisional of application Ser. No. 08/571,785 now U.S. Pat. No. 5,804,411 filed Dec. 13, 1995, which is a divisional of application Ser. No. 08/348,143 filed Nov. 23, 1994 now U.S. Pat. No. 5,506,205.

Priority is claimed under 35 USC 1198 to Japanese patent application Hei-5-315806, filed Nov. 24, 1993.

SUMMARY

The present invention is related to a novel protein p140 polypeptide which is a key protein involved in the signal transmission system of insulin; method for preparation of it; DNA encoding the said polypeptide; vector derived the said DNA; host cells transformed the said vector; antibody of the said polypeptideb pharmaceutical composition containing the said peptide or antibody; method for the prevention and/or treatment of diabetes, which is characterized by tyrosine phosphorylation of the said protein p140 (to be quoted henceforth as phosphorylation in the present detailed specification); agent for the prevention and/or treatment for the currently said the prevention and/or treatment method; agent for the prevention and/or treatment of diabetes, which is characterized by containing a compound which can tyrosine phosphorylate of protein p140, as active ingredient and the screening methods of the said prevention and/or treatment agent.

BACKGROUND OF INVENTION

Diabetes, an abnormal metabolic disease, is induced by a defect in the mechanism of glucose metabolism.

Under normal conditions, glucose metabolism occurs as follows:

Carbohydrates, consumed in t he form of food, are digested to glucose in the intestines prior to absorption into the circulatory system. Pancreatic β cells respond to an increase in the blood glucose level by secreting insulin, which in turn stimulates the target peripheral tissues (muscles and liver) to decrease the blood glucose level by enhancing tissue absorption of the blood glucose followed by conversion to glycogen for storage.

Depending on the causative factors, diabetes is classified into two major categories; insulin dependent diabetes mellitus (IDDM) and non-insulin dependent diabetes mellitus (NIDDM). IDDM (Type I diabetes) is a pathological condition where insulin is not secreted or insufficient even on secretion by pancreatic β cells responding to an increase in the blood glucose level induced by food consumption. It has been known that destruction of β cells of the pancreatic islets induces IDDM. The current therapy employs supplementation of insulin from exogenous sources.

NIDDM (Type II diabetes) is a pathological condition where the feedback mechanism of peripheral tissues is dysfunctional and is ineffective in decreasing the blood glucose level although normal insulin secretion occurs within the living system. In the United States of America, NIDDM is said to be a common disease; 5% of the population exceeding 40 years of age suffer from NIDDM. Causative factors involved in this disease have yet to be elucidated.

RELATED ARTS

Elucidation of the etiology of NIDDM; namely, clarification of the insulin-induced glucose uptake mechanism in peripheral tissue cells is, however, unclear as current knowledge on information transmission mechanism of insulin remains limited and unestablished.

Insulin secreted from the pancreatic islets binds with insulin receptors on the cell membrane of peripheral tissue cells. With regards to post-binding information transmission, the phosphorylase cascade and second messenger theories are the current topics of research.

Briefly, these two theories can be accounted as follows:

Phosohorylase cascade theory:

When insulin binds with the insulin receptor α subunit, the β subunit existing on the inner cell membrane triggers phosphorylation accompanied by activation of the tyrosine kinase site within the receptor. Phosphorylation of substrates by the latter enzyme produces three different proteins. One is composed of 1,235 amino acids and has a molecular weight of 185 kD corresponding to the insulin receptor substrate-1 (IRS-1). On tyrosine phosphorylation of IRS-1, the phosphorylase for phosphatidylinositol, Pl1-kinase, binds against and activates the complex. Post-binding events related to information transmission that concerns localization of glucose transporter within the membrane and membrane ruffling have yet to be established. Other than IRS-1, the existence of two protein substrates (Shc and PTP-1C) has been confirmed. However, the follow-up mechanism(s) has not been completely accounted for.

Second messenger theory:

When insulin binds against the insulin receptor, phospholipase C is specifically activated to degrade phosphatidylinositol glycan (PIG) to produce inositolglycan (IG) and diacylglycerol (DAG) by hydrolysis. Although IG has been reported to display various insulin-like effects, the typical glucose uptake effect has yet to be demonstrated.

However, when protein kinase C is activated by DAG, localization of protein kinase C within the cell membrane has been known to be promoted. This implicates that DAG sequentially phosphorylates inner membrane proteins to finally trigger the glucose uptake. However, this implication remains hitherto unclear.

Although the two different schools of thought have hitherto prevailed, initial stages of the post-binding events related to information transmission can only be explained in part by either theory.

According to Copper et al. in 1988 the hormone, amylin, is released from b pancreatic cells that similar to those that secret insulin when hyperglycemia prevails. Based on their findings that amylin inhibited the action of insulin, they revealed that the hormone might be used as an insulin antagonist. A follow-up report in 1991 indicates that the excessive use of amylin in transgenic mice induces NIDDM. However, the relationship of amylin with insulin information transmission remains hitherto unexplored.

Means to Solve the Problems

The inventors of the present invention focus on the insulin antagonistic properties of amylin. With persistent research activities conducted on the effects of amylin on the insulin information transmission system, the inventors first identified the inhibition site of amylin in regulating the insulin information transmission system and discovered the key proteins, phosphorylated protein 140 and 70 (pp140 and pp70), related to this phenomenon. The present invention reveals clearly the structures of said proteins (DNA base sequences and amino sequences) and elucidation of their functions to totally complement the hitherto deficiently explained insulin information transmission phenomenon.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an effects of vitamin K₅ (VK₅) on blood glucose contents in streptozotocin (STZ)-induced diabetic rats.

FIG. 2 shows an effects of vitamin K₅ (VK₅) on neutral fat contents in blood of streptozotocin (STZ)-induced diabetic rats

FIG. 3 shows an effects of vitamin K₅ (VK₅) on blood cholesterol contents in streptozotocin (STZ)-induced diabetic rats

FIG. 4 shows a hydrophobicity profile for the polypeptide of protein p140 in the present invention

FIG. 5 shows the pUCSRαML2 vector.

DISCLOSE OF THE INVENTION

The present invention related to homologues and fragment sequences of the genuine amino acid sequence of the said protein p140 constructed from SEQ ID No. 1 as shown. In addition, DNAs encoding the related polypeptides of the said homologues and fragment sequences are also encompassed in present invention. Expressed on a more concrete aspect, the said DNAs are those either encoding and/or possessing fragments selectively hybridizing base sequences illustrated in SEQ ID No.2 and 3.

Furthermore, in the present invention, method for the prevention and/or treatment of diabetes, which is characterized by tyrosine phosphorylation of the said protein p140; agent for the prevention and/or treatment for the currently said the prevention and/or treatment method; agent for the prevention and/or treatment of diabetes, which is characterized by containing a compound which can tyrosine phosphorylation of protein p140, as active ingredient and the screening methods of the said prevention and/or treatment agent.

The present invention specifically include:

(1) polypeptides constructed by amino sequence(s) illustrated in SEQ ID No. 1.

(2) DNAs encoding polypeptides described in (1).

(3) DNAs possessing base sequences illustrated in SEQ ID No. 2.

(4) DNAs possessing base sequences illustrated in SEQ ID No. 3.

(5) Method for the prevention and/or treatment of diabetes, which is characterized by tyrosine phosphorylation of protein p140

(6) Agent for the prevention and/or treatment of diabetes, which is characterized by tyrosine phosphorylation of protein p140

(7) Agent for the prevention and/or treatment of diabetes, which is characterized by containing a compound which can tyrosine phosphorylation of protein p140, as active ingredient , and

(8) Method fpr the screening of the agent for the prevention and/or treatment of diabetes, which is characterized by using protein p140.

On administering amylin (0.1 mg/kg, i.p., t.i.d.) to healthy rats for 7 days, dramatic decreases in both incidences of insulin receptor population and secreted insulin quantity. These observations were accompanied by decreases in both incidences, glucose transporter 4 (Glut 4) quantity and synthesized glycogen content (less than 50% decrease compared to that of control group) with 1.7-fold increase in the blood glucose content. Furthermore, in experiments using L6 cells (ATCC strain No., CRL-1458) of rat skeletal muscle myoblasts, a decreased glucose uptake in the cells was observed with amylin administration.

Next, changes in the insulin-induced tyrosine phosphorylation cascade in skeletal muscle myoblasts treated with amylin were investigated by using the anti-phosphotyrosine antibody with the western blot method. As such, when L6 cells were incubated with insulin in the experiments, tyrosine phosphorylation was enhanced. However, pretreatment with amylin under similar conditions confirmed the presence of two different proteins that inhibited the phosphorylation. These proteins are henceforth termed as pp140 and pp70 according to their respective molecular weights. Furthermore, the precursors of these said proteins prior to phosphorylation are, however, henceforth designated as p140 and p70 respectively.

The inventors prepared, isolated and purified the pp140 and pp70 before determining their partial amino acid sequences. On comparing similarities of the said amino acid sequences with previously documented sequences of polypeptides in Swiss Plot Release 2.0, pp70 coincides with the previous known glucose-regulated protein 70. However, the results postulate pp140 as a totally unknown novel protein. As such, inventors of the present invention isolated mRNA of p140 from the rat skeletal muscle myoblasts and constructed the CDNA using the isolated mRNA of p140 before determining the whole base sequence and complete amino acid sequence of the said protein. The results therefore complement the present invention by revealing successfully a completely novel polypeptide and the total DNA chain encoding this polypeptide.

From the above findings, it is understood that amylin may inhibit phosphorylation of p140 and p70 into pp140 and pp70 respectively. In contrast, when amylin is considered to suppress the process from insulin receptor binding to glucose uptake, it suggests that phosphorylation of p140 and p70 to yield pp140and pp70 may play an important role in the glucose uptake mechanism of cells.

The inventors of the present invention attempted to elucidate the mechanism(s) of action of p140 and p70 accordingly.

When rat skeletal muscle myoblasts (rat L6 cells) were incubated in insulin-supplemented cultures, incidence of a pp140 band on day 3 with pp140 production on day 9 were persistently observed. At about the similar interval (day 3), incidence of Glut 4 was similarly observed with gradual increases in rat L6 cell division. Furthermore, polynucleation of rat skeletal muscle myoblasts was observed on day 7 in the similar culture system with subsequent division to form the muscle cells. In the case of pp70,the cells appeared on day 7 and persisted to register production of the protein until day 14.

However, on examining localization of pp140 within the cells, the said protein was found within the microsome membrane (MM) of cytoplasm in the cell at post-culture 10 min when insulin was added to non-serum treated L6 cells. The pp140 disappeared thereafter. In addition, pp140 was first observed in the cell permeable membrane (PM) at post-culture 1˜2 hr. From these findings, pp140 is postulated to have synthesized in cell cytoplasm immediately after insulin treatment ensued with transfer of this protein to permeable membrane (PM) 1˜2 hr thereafter. Furthermore, when pp70 localization in L6 cells was investigated with a similar experimental approach, pp70 was first located in the MM immediately after initiating the culture, registered a peak phosphorylated quantity at post-culture 10 min and gradually approached non-detectable values at post-culture 3 hr. Moreover, pp70 was also located within the nucleus immediately after initiating the culture, and the protein content gradually increased to register a peak value at post-culture 3 hr. From the above protein localization patterns, pp70 exists in MM in the absence of insulin and this protein is mobilized to the nucleus fraction within 3 hr after insulin treatment.

Based on the above results, pp140 information transmission mechanism may be postulated as follows. In short, when insulin binds to the receptor, the latter is activated by auto-phosphorylation. The information is then subjected to undergo various steps of activation via phosphorylation of protein phosphorylases to subsequently phosphorylate p140 to pp140 . The activated pp140 localizes on permeable membrane (PM) surface before p70 is phosphorylated after undergoing various protein phosphorylation processes simultaneously. The phosphorylated pp70 is activated then mobilized to within the nucleus to subsequently trigger biological activities in the Glut 4 expression within the nucleus. Based on this information, Glut 4 produced within the cytoplasm is hence mobilized to localize on the permeable membrane (PM) surface to eventually trigger glucose uptake.

The above information transmission mechanism warrants follow-up experiments to righteously establish concrete evidence of the phenomenon. In any case, it can now be concluded that activation of p140 is an essential step required to induce glucose uptake in cells and subsequent hypoglycemia in the circulatory system.

As such, the present invention is related to method for the prevention and/or treatment of diabetes, especially non-insulin dependent diabetes mellitus (NIDDM), which is characterized by tyrosine phosphorylation of protein p140.

Moreover, the present invention is related to agent for the prevention and/or treatment of diabetes, especially non-insulin dependent diabetes mellitus (NIDDM), which is characterized by tyrosine phosphorylation of protein p140.

In the present invention, method and agent for the prevention and/or treatment of diabetes, which is characterized by tyrosine phosphorylation of protein p140, includes all or whole of the said method and agent for the prevention and/or treatment of diabetes based on the major mechanism of action involving tyrosine phosphorylation of protein p140.

In addition, cells that tyrosine phosphorylate protein p140 are not only confined to skeletal muscle myoblasts (rat L6 cells), but also include all other cells that positively elicits the said phosphorylation. All in all, cells that have been confirmed to display the said phosphorylation include rat FaO hepatocytes, human A673 muscle cells and HepG2 hepatocytes.

Organs other muscles and liver such as the heart, brain, spleen, lungs, kidneys, testes, placenta and pancreas have repeatedly displayed incidences of p140 mRNA of the present invention . Without being confined merely to muscles and liver, the effects of tyrosine phosphorylation may therefore radiate extensively throughout the living system. From this finding, the said mechanism of action of the present invention is hence not limited to muscle and liver cells, but involves the cardiac, encephalic, splenic, pulmonary, renal testical, placental and pancreatic cells as well.

When the polypeptide of the present invention was compared with amino acid sequences of previously known polypeptides recorded with the Swiss Prot Release 2.0, candidates with a complete whole sequence similar to that of the polypeptide was not identified. Furthermore, no a single cDNA of the complete whole polypeptide of the present invention encoding the previously documented nucleotide sequences recorded in the GenBank Release 70 was located. The said peptide of the present invention is hence confirmed to be a completely novel protein.

Additionally, epiterial cell kinase (Eck) and approximately 40% identity were recognized when the results were compared with amino acid sequences of polypeptides previously documented in the Swiss Prot Release 2.0. As such, a novel protein of the present invention was postulated to belong to the Eck family.

In the present invention, a polypeptide of Seq. ID No. 1 in substantially purified form will generally comprise the polypeptide in a production in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the production is that of the Seq. ID No. 1.

A polypeptide homologue of the Seq. ID No. 1 will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the polypeptide of Seq. ID No. 1 over a region of at least 20, preferably at least 30, for instance 40, 60 or 100 more contiguous amino acids. Such polypeptide homologues will be referred to below as a polypeptide according to the invention.

Generally, fragments of Seq. ID No. 1 or its homologues will be at least 10, preferably at least 15, for example 20, 25, 30, 40, 50 or 60 amino acids in length, and are also encompassed by the term “a polypeptide according to the invention” as used herein.

A DNA capable of selectively hybridizing to the DNA of Seq. ID No. 2 or 3 will be generally at least 70%, preferably at least 80 or 90% and more preferably at least 95% homologous to the DNA of Seq. ID No. 2 or 3 over a region of at least 20, preferably at least 30, for instance 40, 60 or 100 or more contiguous nucleotides. Such DNA will be encompassed by the term “DNA according to the invention”.

Fragments of the DNA of Seq. ID No. 2 or 3 will be at least 10, preferably at least 15, for example 20, 25, 30 or 40 nucleotides in length, and are also encompassed by the term “DNA according to the invention” as used herein.

A further embodiment of the invention provides replication and expression vectors comprising DNA according to the invention. The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said DNA and optionally a regulator of the promoter. The vector may contain one or more selectable marker genes, for example a anpicillin resistance gene. The vector may be used in vitro, for example of the production of RNA corresponding to the DNA, or used to transfect or transform a host cell.

A further embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of DNA according to the invention, including the DNA SEQ. ID No. 2 or 3 or the open reading frame thereof. The cells will be chosen to be compatible with the vector and may for example be bacterial, yeast, insect or mammalian.

A further embodiment of the invention provides a method of producing a polypeptide which comprises culturing host cells of the present invention under conditions effective to express a polypeptide of the invention. Preferably, in addition, such a method is carried out under conditions in which the polypeptide of the invention is expressed and then produced from the host cells.

DNA according to the invention may also be inserted into the vectors described above in an antisense orientation in order to proved for the production of antisense RNA. Antisense RNA may also be produced by synthetic means. Such antisense RNA may be used in a method of controlling the levels of a polypeptide of the invention in a cell.

The invention also provides monoclonal or polyclonal antibodies to a polypeptide according to the invention. The invention further provides a process for the production of monoclonal or polyclonal antibodies to the polypeptides of the invention. Monoclonal antibodies may be prepared by conventional hybridoma technology using a polypeptide of the invention or a fragment thereof, as an immunogen. Polyclonal antibodies may also be prepared by conventional means which comprise inoculating a host animal, for example a rat or a rabbit, with a polypeptide of the invention and recovering immune serum.

The present invention also provides pharmaceutical compositions containing a polypeptide of the invention, or an antibody thereof, in association with a pharmaceutically acceptable diluent and/or carrier.

The polypeptide of the present invention includes that which a part of their amino acid sequence is lacking (e.g., a polypeptide comprised of the only essential sequence for revealing a biological activity in an amino acid sequence shown in SEQ ID No. 1), that which a part of their amino acid sequence is replaced by other amino acids (e.g., those replaced by an amino acid having a similar property) and that which other amino acids are added or inserted into a part of their amino acid sequence, as well as those having the amino acid sequence shown in SEQ ID NO. 1.

As known well, there are one to six kinds of codon as that encoding one amino acid (for example, one kind of codon for Met, and six kinds of codon for Leu) are known. Accordingly, the nucleotide sequence of DNA can be changed in order to encode the polypeptide having the same amino acid sequence.

The DNA of the present invention, specified in (2) includes a group of every nucleotide sequences encoding polypeptides (1) shown in SEQ ID NO. 1 . There is a probability of improving a yield of production of a polypeptide by changing a nucleotide sequence.

The DNA specified in (3) is the embodiment of DNA shown in (2), and is sequence in the natural form.

The DNA shown in (4) indicates the sequence of the DNA specified in (3) with a non-translational region.

The DNA having a nucleotide sequence shown in SEQ ID NO. 3 may be prepared according to the following methods, that is:

(i) by isolating mRNA from a cell line which produces the polypeptide of the present invention (e.g., rat skeletal muscle myoblasts L6 cell),

(ii) by preparing first strand (single stranded DNA) from mRNA thus obtained, followed by preparing second strand (double stranded DNA) (synthesis of cDNA),

(iii) by inserting cDNA thus obtained into a proper plasmid vector,

(iv) by transforming host cells with the recombinant DNA thus obtained (preparation of cDNA library),

(v) by random-cloning on a large scale from CDNA library thus obtained, followed by sequencing average 300 bases from 5′ end of each clone, and

(vi) by sequencing complete length of a clone which has a novel base sequence.

Explained in detail, step (i) may be carried out in accordance with the method of Okayama, H. et al. (described in Methods in Enzymology, 154, 3, (1987)) using L6 cells of a rat skeletal muscle myoblasts which is logarithmic growth phase. Examples of the cells which produce the polypeptide of the present invention is rat or human of muscle, liver, heart, brain, spleen, lungs, kidneys, testes, placenta or pancreas, and is preferably rat skeletal muscle myoblasts L6 cell (ATCC strain No., CRL-1458), rat liver FaO cell, human muscle A673 cell or human liver HepG2 cell. Steps (ii), (iii) and (iv) are a series of steps for preparing cDNA library, and may be carried out in accordance with the method of Gubler & Hoffman (Gene, vol. 25, pp. 263, 1983) with a slight modification. As examples of the plasmid vector used in the step (iii), many vectors functioning in an E. coli strain (e.g., pBR 322) and in a Bacillus subtilis (e.g., pUB 110) are known, and pGEM-3Zf(+) (3,199 bp, manufactured by Promega Corp.) which functions in an E. coli, may be preferably used. As examples of host used in the step (iv), many cells are already known. Any cells may be used, and DH5 competent cell which has been prepared in accordance with the method described in Gene, vol. 96, pp. 23, 1990, may be preferably used. The cloning in the step (v) may be carried out by methods known per se and the sequencing may be carried out in accordance with the method of Maxam-Gilbert or the dideoxy termination method. The step (vi) may be carried out in accordance with the method described in Molecular Cloning (written by Sambrook, J., Fritsch, E. F. and Maniatis, T., published by Cold Spring Harbor Laboratory Press in 1989).

As the following step, it is necessary to examine whether or not the DNA thus obtained codes right a produce protein. The examination requires:

(I) the conversion of the DNA sequence into the amino acid sequence in a possible frame,

(II) the confirmation that the DNA thus obtained covers complete or almost complete length of intact mRNA. These confirmation may be carried out after the step (vi) hereinbefore described, and effectively between the step (v) and the step (vi).

The step (II) may be carried out by Northern analysis.

Once the nucleotide sequences shown in SEQ ID NOs. 2 and 3 are determined, DNA of the present invention may be obtained by chemical synthesis, by PCR method or by hybridization making use of a fragment of DNA of the present invention, as a probe. Furthermore, DNA of the present invention may be obtained in a desired amount by transforming with a vector DNA inserted a DNA of the present invention into a proper host, followed by culturing the transformant.

The polypeptides of the present invention (shown in SEQ ID NO. 1) may be prepared by:

(1) isolating and purifying from an organism or a cultured cell,

(2) chemically synthesizing, or

(3) using a skill of biotechnology, preferably, by the method described in (3).

Examples of expression system when preparing a polypeptide by using a skill of biotechnology is, for example, the expression system of bacteria, yeast, insect cell and mammalian cell.

For example, the expression in E. coli may be carried out by adding the initiation codon (ATG) to 5′ end of a DNA encoding a nucleotide sequence shown in SEQ ID NO. 3, connecting the DNA thus obtained to the downstream of a proper promoter (e.g., trp promoter, lac promoter, λ_(PL) promoter, T7 promoter etc.), and then inserting it into a vector (e.g., pBR322, pUC18, pUC19 etc.) which functions in an E. coli strain to prepare an expression vector. Then, an E. coli strain (e.g., E. coli DH1 strain, E. coli JM109 strain, E. coli HB101 strain, etc.) which is transformed with the expression vector thus obtained may be cultured in a proper medium to obtain the desired polypeptide. When a signal peptide of bacteria (e.g., signal peptide of pel B) is utilized, the desired polypeptide may be also secreted in periplasm. Furthermore, a fusion protein with other polypeptide may be also produced easily.

Furthermore, the expression in a mammalian cell may be carried out, for example, by inserting the DNA shown in SEQ ID NO. 3 into the downstream of a proper promoter (e.g., SV40 promoter, LTR promoter, metallothionein promoter etc.) in a proper vector (e.g., retrovirus vector, papilloma virus vector, vaccinia virus vector, SV40 vector, etc.) to obtain an expression vector, and transforming a proper mammalian cell (e.g., monkey COS-7 cell, Chinese hamster CHO cell, mouse L cell etc.) with the expression vector thus obtained, and then culturing the transformant in a proper medium to get a desired polypeptide in the culture medium. The polypeptide thus obtained may be isolated and purified by conventional biochemical methods.

The protein of the present invention includes the reaction products of phosphorylated and/or sugar-chained protein. In short, the present invention contains p140-bound polysaccharide chains and tyrosine phosphorylated p140 (pp140 ) found in p140 polypeptides.

EFFECTS OF INVENTION

The protein p140 is postulated to possess the above-mentioned mechanism of action. The protein p140 polypeptide of the present invention can therefore not only improve the hyperglycemic conditions when used alone, but can also be useful in prevention and/or treatment for diabetes, especially non-insulin dependent diabetes mellitus (NIDDM).

Further, polyclonal or monoclonal antibody against the protein p140 polypeptide of the present invention can be used in the determination of the amount of the said polypeptide in organism, and thereby, may be utilized for the purpose of investigating the relationship between the said polypeptide and diseases, or for the purpose of diagnosing diseases, and the like. Polyclonal and monoclonal antibody thereof may be prepared by conventional methods by using the said polypeptide or the fragment thereof as an antigen.

The DNA of the present invention may be utilized as an important and essential template in preparing the polypeptide of the present invention which is expected to possess various use or for diagnosis of and in the treatment of gene diseases (the treatment of gene defect disease and the treatment by inhibiting expression of the polypeptide by antisense DNA (RNA), and the like). Further, genomic DNA may be isolated by using the DNA of the present invention as a probe. Similarly, it is possible to isolate genes having high homology to the DNA of the present invention in human or those of other species.

Furthermore, the present invention is related to agent for the prevention and/or treatment of diabetes characterized by containing a compound which can tyrosine phosphorylation of protein p140, as active ingredient.

All in all, tyrosine phosphorylated protein p140 products include not only currently confirmed substances that possess the said activities but also all those substances that will be confirmed to possess the said activities henceforth. At present, it is confirmed that the compounds have activity of tyrosine phosphorylation, for example,

(1) the benzene or naphthalene derivatives of the formula (I)

wherein R¹ of n species each, independently, is hydrogen atom C1-4 alkyl, hydroxy, amino or COOR² (in which R² is hydrogen atom or C1-4 alkyl), n is 1-3 and non-toxic salts thereof and non-toxic acid addition salts thereof,

(2) the benzoquinone or naphthoquinone derivatives of the formula (II)

wherein R³ of m species each, independently, is hydrogen atom, C1-12 alkyl, 1-4 alkoxy, C1-4 alkylthio, hydroxy, halogen, phenyl or phenyl substituted by halogen, m is 1-4,

(3) the rhodanine or thazolidine derivatives of the formula (III)

wherein X is oxygen or sulfur atom, R⁴ and R⁵ each, independently, is hydrogen atom, phenyl or phenyl substituted by C1-4 alkyl, C1-8 alkoxy, halogen atom or nitro, or R⁴ and R⁵, taken together, represent benzylidene, benzylidene substituted by C1-4 alkyl, C1-8 alkoxy, halogen atom or nitro or β-methylcinnamilidene, R⁶ is hydrogen atom C1-4 alkyl, and non-toxic salts thereof and non-toxic acid-addition salts thereof.

More concretely, the compounds of the formula (I) include 4-amino-2-hydroxybenzoic acid, 4-amino-1-naphthol, 4-amino-2-naphthol, 1-aminonaphthalene, 1,4-dihydroxynaphthalene, 4-amino-2-methyl-1-naphthol (abbreviated as vitamin K₅ hereinafter), 1,4-dihydroxy-2-naphthenic acid, etc.

The compounds of the formula (II) include 2-methyl-1,4-benzoquinone, 2,6-di-tert-butyl-1,4-benzoquinone, 2,6-dibromo-1,4-benzoquinone, 2,3,4,5-tetrafluoro- 1,4-benzoquinone, 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone (abbreviated as vitamin K3 hereinafter), 2-hydroxy-3-methyl-1,4-naphthoquinone, 2-(3,7-dimethyloctyl)-3-hydroxy-1,4-naphthoqunone, 2-methoxy-3-methyl-1,4-naphthoquinone, 2-hydroxy-1,4-naphthoquinone, 3-(4-chlorophenyl)-2-hydroxy-1,4-naphthoquinone, 2-propylthio-1,4-naphthoquinone, etc.

The compounds of the formula (III) include 5-phenylrhodanine, 5-phenyl-1,3-thiazodidine-2,4-dione, 5-benzylidenerhodanine, 5-benzylidene-1,3-thiazodidine-2,4-dione, 5,5-diphenylrhodanine, 5,5-diphenyl- 1,3-thiazodidine-2,4-dione, 5-(4-isoamyloxybenzylidene)rhodanine, 5-(4-isoamyloxybenzylidene)-1,3-thiazodidine-2,4-diene, 5-(β-methylcinnamylidene)rhodanine-3-acetic acid, etc., and non-toxic salts thereof and non-toxic acid addition salts thereof.

In the present invention, the appropriate non-toxic salts, for example, are salts of alkali metal (e.g., potassium, sodium etc.), salts of alkaline earth metal (e.g., calcium, magnesium etc.), ammonium salts, salts of pharmaceutically-acceptable organic amine (e.g., tetramethylammonium, triethylamine, methylamine, dimethylamine, cyclopentylamine, benzylamine, phenethylamine, piperidine, monoethanolamine, diethanolamine, tris(hydroxymethyl)amine, lysine, arginine, N-methyl-D-glucamine etc.).

In the present invention, the appropriate acid addition salts include the salts with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and nitric acid, and the salts with organic acids such as acetic acid, trifluoroacetic acid, lactic acid, tartaric acid, oxalic acid, fumaric acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid, isethionic acid, glucuronic acid and gluconic acid.

The compound of the formulae (I), (II) and (III) are well known per se, or used as other starting materials are may be easily prepared by methods known perse.

As the substances used in the present invention are subjected to tyrosine phosphorylation, these agents not only improve the diabetes-derived hyperglycemic conditions but are also useful for the treatment and/or prevention of diabetes, especially non-insulin dependent diabetes mellitus (NIDDM).

It was confirmed that the toxicity of the various active ingredient and salts thereof of the present invention is very low. Therefore, it may be considered that the various active ingredient and acid-addition salts thereof of the present invention are safe and suitable for pharmaceutical use.

For the purpose above described, the polypeptide, each active ingredient and acid addition salts thereof of the present invention, may be normally administered systemically or partially, usually by oral or parenteral administration.

The doses to be administered are determined depending upon e.g., age, body weight, symptom, the desired therapeutic effect, the route of administration, and the duration of the treatment. In the human adult, the doses per person per dose are generally between 10 μg and 1000 mg, by oral administration, up to several times per day, and between 10 μg and 100 mg, by parenteral administration up to several times per day, or by continuous intravenous administration between 1 and 24 hrs. per day.

As mentioned above, the doses to be used depend upon various conditions. Therefore, there are cases in which doses lower than or greater than the ranges specified above may be used.

When administration of the compounds of the present invention, it is used as solid compositions, liquid compositions or other compositions for oral administration, as injections, liniments or suppositories for parenteral administration.

Solid compositions for oral administration include compressed tablets, pills, capsules, dispersible powders, and granules. Capsules include hard capsules and soft capsules.

In such compositions, one or more of the active compound(s) is or are admixed with at least one inert diluent (such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch, polyvinylpyrrolidone, magnesium metasilicate aluminate). The compositions may also comprise, as is normal practice, additional substances other than inert diluents: e.g. lubricating agents (such as magnesium stearate), disintegrating agents (such as cellulose calcium glycolate), stabilizing agents (such as lactose), and agents to assist dissolution (such as glutamic acid, asparaginic acid).

The tablets or pills may, if desired, be coated with a film of gastric or enteric material (such as sugar, gelatin, hydroxypropyl cellulose or hydroxypropylmethyl cellulose phthalate), or be coated with more than two films. Coating may include containment within capsules of absorbable materials such as gelatin.

Liquid compositions for oral administration include pharmaceutically-acceptable solutions, emulsions, suspensions, syrups and elixirs. In such compositions, one or more of the active compound(s) is or are contained in inert diluent(s) commonly used in the art (such as purified water, ethanol). Besides inert diluents, such compositions may also comprise adjuvants (such as wetting agents, suspending agents), sweetening agents, flavouring agents, perfuming agents, and preserving agents.

Other compositions for oral administration include spray compositions which may be prepared by known methods and which comprise one or more of the active compound(s). Spray compositions may comprise additional substances other than inert diluents: e.g. stabilizing agents (such as sodium sulfate), isotonic buffer (such as sodium chloride, sodium citrate, citric acid).

For preparation of such spray compositions, for example, the method described in the U.S. Pat. No. 2,868,691 or 3,095,355 may be used.

Injections for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. In such compositions, one more active compound(s) is or are admixed with at least one inert aqueous diluent(s) (such as distilled water for injection, physiological salt solution) or inert non-aqueous diluent(s) (such as propylene glycol, polyethylene glycol, olive oil, ethanol, POLYSORBATE 80 (registered trade mark)).

Injections may comprise additional materials other than inert diluents: e.g. preserving agents, wetting agents, emulsifying agents, dispersing agents, stabilizing agent (such as lactose), and agents to assist dissolution (such as glutamic acid, asparaginic acid).

They may be sterilized for example, by filtration through a bacteria-retaining filter, by incorporation of sterilizing agents in the compositions or by irradiation. They may also be manufactured in the form of sterile solid compositions, for example, by freeze-drying, and which may be dissolved in sterile water or some other sterile diluent(s) for injection immediately before used.

Other compositions for parenteral administration include liquids for external use, and endermic liniments, ointment, suppositories and pessaries which comprise one or more of the active compound(s) and may be prepared by methods known per se.

EXAMPLE

The following examples are illustrated, but not limit, the present invention.

Example 1 Purification Method of pp140

By employing cell trays (225 cm²), rat L6 cells were incubated at 37° C. for 7˜10 days in 5% CO₂ atmosphere. Culture media were replaced at 3-day intervals with the Dulbecco's modified Eagle's medium (containing 10% bovine fetal serum (BFS)). Two hours after treating the muscle cells developed from skeletal muscle myoblasts with serum-free medium, 500 μM vanadic acid (vanadate) was added to the culture and allowed to incubate further for 10 min. Cells were then suspended in Tris buffer (400 μM vanadate with protease inhibitor), lysed and centrifuged prior to isolating the supernatant.

The supernatant was adjusted with octa (ethylene glycol) ether (C₁₂E₈) to a final concentration of 0.1% before filtration through a millipore membrane. Protein G sepharose gel bound with anti-phosphotyrosine antibodies was filled with the filtered sample. The tyrosine phosphorylated protein (pp140) adsorbed to the gel. After rinsing the column with 25 mM Tris buffer, pp140 was eluted with 10 mM phenylphosphate. The eluate was concentrated with Centricon 30 prior to precipitating pp140 by the acetone precipitation method.

Example 2 Tyrosine Phosphorylation of p140 in Various Tissues

Using the Dubecco's modified Eagle's medium (containing 10% BFS), various cells (1×10⁵ cells/dish) were incubated at 37° C. under 5% CO₂ atmosphere for 5˜8 days. The cells were skeletal muscle cells differentiated from skeletal muscle myoblasts. The differentiated cells previously treated in serum-free Dulbecco's modified Eagle's medium for 4 hr was incubated with and without amylin (100 pM) before further incubation for 24 hr. Cultures treated with insulin (100 nM) thereafter were incubated for a fixed interval (10 or 60 min).

After the cultures were rinsed with ice-cold phosphate buffer, cells were lysed with phosphate buffer containing 0.5% octa (ethylene glycol) ether (C₁₂E₈). The pp140 was recovered by sepharobeads bound with phosphothyrosine antibody (Transformation Corp.) prior to elution and detection with phenyl phosphate and western blotting method, respectively. The band content of pp140 was determined by a densitometer using purified pp140 as the standard. The results are illustrated in Table 1.

TABLE 1 Effects of p140 tyrosine phosphorylation on various tissues rat human L6 FaO A 678 HepG2 control  300  100  300  250 insulin 10 min 2400 2000 2500 2100 insulin 60 min 1000 1400 1900 1200 insulin added amylin 10 min  180  300  350  300 insulin added amylin 60 min  200  100 1000  300

In the Table 1, cultures were treated with amylin (100 pM) 24 hr before insulin (100 nM) was added.

Observation

Incidence of pp140 , observed when rat L6 cells were incubated with insulin within 10 min, was antagonized by amylin treatment. Moreover, this phenomenon was similarly confirmed in rat hepatocytes, FaO cells. Furthermore, this phenomenon is not merely confined to rats. In human muscle cells (A673 cells) and hepatocytes (HepG2 cells), the phenomenon has been similarly confirmed. It is postulated that amylin suppresses a certain stage or processes before p140 phosphorylation is triggered by the phosphorylation signal of insulin.

Example 3 Effects of Various Test Compounds on p140 Phosphorylation

Rat L6 cells (1×10⁵ cells/dish) were incubated in the Dulbecco's modified Eagle's medium (containing 10% BFS) at 37° C. under 5% CO₂ atmosphere for 8 days. The cells used were skeletal muscle myoblast-differentiated muscle cells. After treating the differentiated skeletal muscle cells in serum-free Dulbecco's modified Eagle's media for 4 hr, various test compounds (10 mM; except insulin, 1 mM) were added before the cultures were further incubated for a fixed interval.

After the cultures were rinsed with ice-cold phosphate buffer, cells were lysed with phosphate buffer containing 0.5% octa (ethylene glycol) ether (C₁₂E₈). The pp140 was recovered by cephalobeads bound with phosphotyrosine antibody (Transformation Corp.) prior to elution and detection with phenyl phosphate and western blotting method, respectively. The band content of pp140 was determined by a densitometer using purified pp140 as the standard. The results are illustrated in Table 2.

TABLE 2 Effects of p140 tyrosine phosphorylation of various test compounds Amount of tyrosine phosphorylated p140 (copy/cell) Compound 0 3 10 60 (min) Vitamin K₃ 350 3650 1800  750 Vitamin K₅ 400 3850 2850 1600 5-phenylrhodanine 350 1600 1250  650 5-benzylidenerhodanine 400 2650 1900 1350 5-(4-isoamyloxybenzylidene) 400 3200 2250 1600 rhodanine Insulin 350 1850 2600 1650 (positive control)

Example 4 Enhancement Activity on Glucose Uptake

Rat L6 cells (1×10⁵ cells/dish) were incubated in Dulbecco's modified Eagle's medium (containing 10% BFS) at 37° C. under 5% CO₂ atmosphere for 8 days. The cells used were skeletal muscle myoblast-differentiated skeletal muscle cells. After treating the differentiated skeletal muscle cells in serum-free Dulbecco's modified Eagle's medium for 2 hr, various test compounds (10 mM; except insulin, 1 mM) were added before the cultures were further incubated for a fixed interval of 2 hr. Cultures thereafter treated with Crebs-Ringer phosphate buffer (pH: 7.4) for 20 min were further incubated with 5 mM ³H-2-deoxyglucose (0.05 mCi/ml). At the initial 3 min after incubation, the uptake radioactivity content in cells was determined with a liquid syntillation counter. The results are illustrated in Table 3.

TABLE 3 Enhancement activity on glucose uptake Activity on glucose uptake Compound (pmol/mg protein/min) Control 22.6 Vitamin K₃ 60.9 Vitamin K₅ 67.5 5-phenylrhodanine 76.8 5-benzylidenerhodanine 84.2 5-(4-isoamyloxybenzylidene) 98.4 rhodanine Insulin 106.8 (positive control)

Observation

All compounds that promoted p140 phosphorylation were confirmed to activate glucose uptake activities (Table 2 and 3).

Example 5 Effects of Vitamin K₅ on Diabetes

The diabetes model using streptozotocin (STZ) was established in male Wistar rats (STZ rats). After administering various intraperitoneal (i.p.) daily doses of vitamin K₅ for 3 consecutive days in STZ rats (one administration per day), the glucose, neutral fat and cholesterol contents in blood were determined. Accordingly, STZ and normal rats were administered with the vehicle (physiological saline) at identical daily rate and duration prior to determination of similar hematic indices mentioned above. In addition, rats administered subcutaneously (s.c.) with insulin (8 U/kg) daily (one administration per day) for 3 consecutive days were used as positive controls. The results are shown in FIG. 1 to 3.

Observations

Administration with vitamin K₅ for 3 consecutive days elicited recovery of changes found in all hematic indices in rats; namely, the glucose, neutral fat and cholesterol contents.

Example 6 Analysis of Partial Amino Acid Sequence of pp140

pp140 purified in Example 1 was isolated by electrophoresis, followed by transcription in PVDF membrane, treatmented with trypsin and further isolated with liquid chromatography. The thus isolated pp140 fragment was then sequenced by using the 470A-model automated gas-phase protein sequences/120A-model PTH analyzer (ABI or Applied Biosystem Inc. Corp., U.S.A.) and the extensively employed Edman degradation method prior to determination of its partial amino acid sequence. The sequence is depicted in Sequence Table 5 to 7.

Example 7 Partial Amino Acid Sequencing of pp140 by the Polymerase Chain Reaction (PCR) Method

By using extensively applied methods, various primers were derived from the thus isolated partial amino acid fragments, and their respective combinations were conducted before the PCR method was employed. The results revealed a specifically amplified fragment with an approximate length of 400 bp.

Example 8 Isolation and Purification of mRNA

During the log growth phase, mRNA was isolated from 3×10⁷ muscle myoblast L6 cells (ATCC strain No., CRL-1458) according to the method of Okayama et al (Methods in Enzymology, 154, 3 (1987)).

Briefly, after cells were lysed with 5.5 M GTC solution (5.5 M guanidine thiocyanate, 25 mM sodium citrate and 0.5% sodium lauryl sarcosine, the lysate was layered on cesium trifluoroacetate solution (density: 1.51) cells lysate and centrifuged at 120,000×g for 20 hr before all the RNA in the pellet was recovered. The RNA sample was passaged through an oligo-dT-cellulose column twice prior to recovery by purification of 106 μg poly(A)⁺RNA.

Example 9 Tissue distribution of p140 mRNA

From various tissues, poly(A)⁺RNA was purified according to procedures similar to those of Example 8. The respective tissue-derived poly(A)⁺RNA samples (each sample: 2 μg) were subjected to agarose-gel electrophoresis and subsequently transferred through a filter. The 2-kb open reading frame was labeled and used as the internal control before allowed to undergo normal hybridization. Autoradiography was conducted on the specifically bound probe and evaluated by densitometric analyses with an imaging analyzer. When the incidence of β-actin mRNA was taken 100 in the various tissues, relative contents of p140 in tissues are indicated in Table 4.

TABLE 4 Tissue distribution of p140 mRNA rat human heart 100 100 brain 240  60 spleen  70 — lungs 210 100 liver 130 100 muscles  40 130 kidneys 130  40 testes, 320 — placenta — 220 pancreas — 330 — represents experiments that were not done

Observation

Examination of all the various tissues studied reveals incidences of mRNA, whose effects are though to radiate over an extensive range of tissues. High incidence of mRNA is found especially in the human pancreas.

Example 10 Establishing the cDNA Library

A cDNA library was established according to the modified Gubler and Hoffman method (Gene 25, 263, (1983)).

From poly(A)⁺RNA (5 μg) derived in Example 2, a first strand was constructed with the reverse transcription enzyme, followed by transformation of a second strand with EcoRI adaptor ligation before excess adaptors and primers were eliminated by gel filtration column chromatography (Sephacryl S-500HR column; Pharmacia Corp.). The remaining 1,620 ng of cDNA fraction was subsequently recovered.

The above construction procedures for cDNA library were accomplished with a λgt 10 cloning system kit (Amersham Corp.).

Next, the λgt 10 phage (Amersham Corp.) and λZAPII phage (Stra Tagene Corp.) were ligated at the EcoRI-treated arms of 1.8-kb mean length. A phage cDNA library of an independent count of 3×10⁵ was established.

Example 11 Cloning and Sequencing

Based on the phage DNA library established in Example 10, clones were duplicated to approximately 1×10⁵ plagues/plate. The approximately 400-bp fragments harvested in Example 7 were designated as probes before screening was conducted. Of the positive controls, subcloning of long strands of the inserts in EcoRI side of plasmid vector pGEM-3Zf(+) (3199 bp; Promega Corp.) was established. T7 or SP6 was sequenced as the primer.

DNA sequencing based on the dideoxy terminator method was performed according to the cyclo-sequencing method using fluorescent di-terminator (ABI, USA). Furthermore, sequence reading was realized with a DNA sequencer (Model 373A; ABI, USA).

As such, nucleotide sequences of mean 300 bases were established from 5′ or 3′ side of the respective clone.

Example 12 Partial Sequence Analysis

When the nucleotide sequence from Example 11 was subjected to a homology search with all the nucleotide sequences stored in previously registered data base (GenBank and EMBL) with the FASTA program of Lipman and Pearson, the sequenced clones would identify clones containing novel sequences. Nucleotide sequences of the identified clone were converted to amino acid sequences based on 3 possibly constructed frames.

Additionally, novel amino acid sequences in the amino acid sequences were also revealed.

However, the cDNA clone that has cloned does not necessarily cover the whole mRNA length. In such a case, the clone is most unlikely to contain the N terminal of amino acid sequence.

As such, the Northern analysis was used to determine if the whole length of the established clone was complemented. In other words, the poly(A)⁺RNA, isolated from Example 8→Example 9 procedures by electrophoresis, was blotted on a nylon membrane. When the subcloned cDNA insert was hybridized as a probe, a single band at approximately 4400-bp position was observed. Since sizes of the clones were approximated to 2200 bp, PCR was performed at the 5′ and 3′ sides to read the whole cDNA length with the 3′-RACE (BRL Corp.) system and 5′-RACE (CLONTECH Corp.) system kits.

Example 13 Determining the Sequence and Open Reading Frame of Whole cDNA Length

Random sequencing of the whole length of cDNA sequence was appropriated according to the method of Sambrook et al. (Molecular Cloning: ed. Sambrook J, Fritsch E F, Maniatis T; 1989, Cold Spring Harbor Laboratory Press).

Briefly, plasmid was recovered from the clone and the isolated cDNA insert was then purified before ligation and fragmentation. The terminal of DNA fragment was further smoothened by T4 polymerase and DNA fragments of approximated 400-bp length were recovered by agarose electrophoresis. DNA fragments thus established were subjected to cloning in the Smal side of plasmid vector and pGEM-3Zf(+) (3199 bp; Promega Corp.) before transformation in E.Coli. Eighty colonies were picked up at random and plasmid DNAs were refined prior to DNA sequencing of these 20 plasmids (possessing cDNA fragments as inserts). DNA sequencing and sequence reading were performed according to the method described in Example 11. Sequence data of cDNA fragments were constructed to the linkage sequences with the DNA sequence program of DNASIS. The basic sequence portrayed in Seq. ID No 3 was hence constructed. From sequence data of the whole cDNA length, the open reading frame (ORF) was determined. The amino acid sequence was further translated and the sequence thus established is illustrated in Seq. No 1. One of the frames possesses the 2993-bp ORF, that was approximated to 3,000 bp of the whole ORF length of the Eck family. Therefore, the said polypeptide in the present invention is postulated to possess a whole length of 2,993 bp.

Based on its hydrophobicity, protein p140 was further postulated to be a typical Type I membrane protein (FIG. 4 demarcates the zone with either high (+) or low (−) hydrophobicity).

All in all, the said p140 polypeptide is a typical membrane protein with 993 amino acids and the length of its ORF is 2982 bp. Furthermore, the estimated molecular weight of the said p140 polypeptide is 109,860 Da, and is 140 kD when evaluated from the bonds of its polysaccharide chain.

Example 14 Construction of Plasmid Vector for Using the Preparation of Expression Vector

As an expression vector, pUC-SRαML-1 (This vector is disclosed itself and preparation thereof in European Patent publication No. 559428) derivative was used. This derivative was constructed to insert two kinds of fragments as shown below:

fragment T7 5′ GTAATACGACTCACTATAGGGGAGAGCT 3′ (SEQ ID No. 8) 3′  ACGTCATTATGCTGAGTGATATCCCCTC 5′ (SEQ ID No. 9) between Pstl and Sacl and fragment SP6 5′ CTAGTCTATAGTGTCACCTAAATCGTGGGTAC 3′ (SEQ ID No. 10) 3′ AGATATCACAGTGGATTTAGCAC 5′ (SEQ ID No. 11)

between Spel and Kpnl site in the multi-cloning site, respectively.

The pUC-SRαML1 vector was digested with Pstl and Sacl and the resulting digest was subjected to agarose gel electrophoresis to prepare and recover an about 4.1 kbp fragment and thereafter removing the 5′-end phosphoric acid group by BAP (bacterial alkaline phosphatase) treatment. The phosphorylated DNA fragment T7 was ligated with the thus prepared about 4.1 kbp fragment from pUC-SRαML1 to make them into a circular form. The resulting vector was, moreover, digested with Spel and Kpnl and the resulting digest was subjected to agarose gel electrophoresis to prepare and recover an about 4.1 kbp fragment and thereafter removing the 5′-end phosphoric acid group by BAP (bacterial alkaline phosphatase) treatment. The phosphorylated DNA fragment SP6 was ligated with the thus prepared about 4.1 kbp fragment to make them into a circular form. The plasmid vector constructed in this manner was named pUC-SR═ML2 (See FIG. 3).

Example 15 Construction of Expression Vector

The primers X, Y and YH, that aneal to rat p140 cDNA, were synthesized. Sequences of primers X, Y and YH are as follows:

Primer X

5′—A ATA TAG TCG ACC ACC ATG GAG AAC CCC TAC GTT GGG CGA GCG A—3′

(SEQ ID No. 12)

Primer Y

5′—CGG CGG ACT AGT TCA GAC CTG CAC GGG CAG TGT CTG G—3′

(SEQ ID No. 13)

Primer YH

5′- GCC GCC ACT AGT TCA GTG GTG GTG GTG GTG GTG GAC CTG CAC GGG CAG TGT CTG G—3′

(SEQ ID No. 14)

The plasmid containing cDNA of p140 was subjected to PCR using the thus synthesized oligonucleotides X and Y as templates. The thus obtained PCR fragment contains a sequence placed 5′-adjacent to the initiation codon, that is corresponding to Cozac sequence which is known among skilled in the art, and cDNA which encodes a protein molecule consisting of the p140 protein. The PCR fragment was digested with SalI-Spel and the resulting digest was separated and purified and then inserted into the SalI-Spel site of the pUC-SRαML2 prepared in Example 14 to obtain an expression vector pUC-SRαML2-p140-A.

Moreover, the plasmid containing cDNA of p140 was subjected to PCR using the synthesized oligonucleotides X and YH as templates. The thus obtained PCR fragment contains a sequence placed 5′-adjacent to the initiation codon, that is corresponding to Cozac sequence which is known among skilled in the art, and cDNA which encodes a protein molecule consisting of the p140 protein and six additional histidine (His) residues attached to its C-terminal end. The PCR fragment was digested with SalI-Spel and the resulting digest was separated and purified and then inserted into the SalI-Spel site of the pUC-SRαML2 prepared in Example 14 to obtain an expression vector pUC-SRαML2-p140-B.

Moreover, primer Z and ZH were synthesized. Sequences of primer Z and ZH are as follows: (These were adjoined to amino-terminal end of transmembrane region in cDNA.)

Primer Z

5′—CGG CGG ACT AGT TCA TGA GCC TCT TTC ACT CGT GGT CTC AAA CT—3′

(SEQ ID No. 15)

Primer ZH

5′—GCC GCC ACT AGT TCA GTG GTG GTG GTG GTG GTG TGA GCC TCT TTC ACT CGT GGT CTC AAA CT—3′

(SEQ ID No. 16)

The plasmid containing cDNA of p140 was subjected to PCR using the thus synthesized oligonucleotides X and Z as templates. The thus obtained PCR fragment contains a sequence placed 5′-adjacent to the initiation codon, that is corresponding to Cozac sequence which is known among skilled in the art, and cDNA which encodes a polypeptide consisting of the p140 protein extracellular part. The PCR fragment was digested with SalI and Notl and the resulting digest was separated and purified and then inserted into the SalI-Spel site of the pUC-SRαML2 prepared in Example 14 to obtain an expression vector pUC-SRαML2-p140-C.

Moreover, the plasmid containing cDNA of p140 was subjected to PCR using the synthesized oligonucleotides X and ZH as templates. The thus obtained PCR fragment contains a sequence placed 5′-adjacent to the initiation codon, that is corresponding to Cozac sequence which is known among skilled in the art, and cDNA which encodes a polypeptide consisting of the p140 protein extracellular part and six additional histidine (His) residues attached to its C-terminal end. The PCR fragment was digested with SalI-Spel and the resulting digest was separated and purified and then inserted into the SalI-Spel site of the pUC-SRαML2 prepared in Example 14 to obtain an expression vector pUC-SRαML2-p140-D.

Each of the thus constructed pUC-SRαML2-p140-A, pUC-SRαML2-p140-B, pUC-SRαML2-p140-C and pUC-SRαML2-p140-D plasmids were transfected into an E. coli strain DH5, recovered from a 100 ml culture of the resulting transformant and then purified by CsCl density gradient centrifugation twice.

Example 16 Expression in COS Cells

Each of the plasmid DNA preparations pUC-SRαML2, pUC-SRαML2-p140-A, pUC-SRαML2-p140-B, pUC-SRαML2-p140-C and pUC-SRαML2-p140-D were introduced into COS-7 cells (Cell, 23, 175 (1981)) by means of the diethylaminoethyl (DEAE) dextran method (J. Immunology, 136, 4291 (1986)).

That is, about 1.8×10⁶ COS-7 cells were inoculated into a 225 cm² capacity flask (manufactured by Corning) together with 50 ml of a liquid culture medium (Dulbecco's modified MEM medium supplemented with 10% decomplemented fetal bovine serum). After overnight incubation in a carbon dioxide incubator (37° C., 5% CO₂) and subsequent removal of the culture supernatant, 12 ml of a DNA cocktail (Dulbecco's modified MEM medium supplemented with 15 μg of each plasmid DNA, 50 mM Tris-HCl buffer (pH 7.4) and 400 μg/ml of DEAE-dextran) was added to each flask and culture was carried out for 3 hours at 37° C. in an atmosphere of 5% CO₂. Thereafter, the DNA cocktail was replaced by 15 ml of a chloroquine solution (Dulbecco's modified MEM medium supplemented with 150 μM chloroquine and 7% decomplemented fetal bovine serum), followed by additional 3 hours of culture.

After removing the chloroquine solution, the aforementioned liquid culture medium (50 ml) was added to each of the resulting flasks which were then incubated at 37 OC in an atmosphere of 5% CO₂ for 72 hours to find growth of the cells in each flask into almost monolayer form. After removing the culture supernatant, the cells in each flask were washed with a serum-free liquid culture medium (trade name, SFM-101; available from Nissui Pharmaceutical Co., Ltd.) and then supplied with 75 ml of the same serum-free liquid culture medium, and the culturing was continued for another 72 hours. Thereafter, the resulting culture supernatants were recovered and cells were lysed as represented in Example 1. These supernatants and cell lysates were filtered through a membrane filter (trade name, STERIVEX-GS; available from Millipore Corp.) to remove cell debris. The thus obtained culture supernatant samples were stored at 4° C. for future use. A The cell lysates of COS cells which have been transformed with plasmid containing the pUC-SRαML2-p140-A and pUC-SRαML2-p140-B inserts are expected to contain expressed mature protein moieties of polypeptides which correspond to p140 protein. And culture supernatants of COS cells which have been transformed with plasmid containing the pUC-SRαML2-p140-C and pUC-SRαML2-p140-D inserts are expected to contain secreted polypeptides which correspond to p140 protein extracellular part.

Example 17 Confirmation of Expression

A 2 ml portion of each of the culture supernatants of transformed COS cells obtained in Example 16 was concentrated to a volume of 100 ml using a centrifugal concentration filter (trade name, Centricon-10; available from Millipore Corp.). A 1 μl portion of each of the thus concentrated samples was mixed with the same volume of a loading buffer (0.125 M Tris-HCl buffer (pH 6.8), 4% sodium dodecyl sulfate and 30% glycerol) for SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) use, and the mixture was treated at 90° C. for 3 minutes and then subjected to SDS-PAGE.

In the case of the pUC-SRαML2-p140-B and pUC-SRαML2-p140-D proteins having His hexamer introduced to the C-terminus of the proteins, not only their corresponding cell lysates and COS cell culture supernatants but also their purified products were subjected to the SDS-PAGE analysis.

Purification of the protein was carried out by means of a metal chelate affinity chromatography (Biotechnology, 9, 273, (1991)), making use of the function of His to form complex compounds with various transition metal ions. That is, a culture supernatant (350 ml) or cell lysates (100 ml) obtained from COS cells was mixed with a sodium chloride aqueous solution in such an amount that the final concentration of the salt became 1 M, and the resulting mixture was applied to a column packed with 4 ml of a zinc-linked chelating Sepharose (trade name, Chelating Sepharose Fast-Flow; available from Pharmacia) to adsorb the protein to the resin. The column was washed with 50 mM phosphate buffer (pH 7.0) containing 1 M sodium chloride aqueous solution (40 ml), and the protein retained in the column was eluted with 50 mM phosphate buffer (pH 7.0) containing 1 M sodium chloride aqueous solution and 0.4 M imidazole. Thereafter, the resulting elute was concentrated to a volume of 100 μl, and a portion of the concentrated sample was subjected to SDS-PAGE analysis.

The SDS-PAGE analysis was carried out using a SDS 10/20 gradient gel and a product which corresponds to a molecular weight of p140 was detected in samples prepared from COS cells transfected pUC-SRαML2-p140-A and p140-B. Furthermore, a polypeptide which corresponds to a molecular weight of extracellular portion of p140 was detected in untreated and purified supernatants, not cell lysates, prepared from COS cells transfected pUC-SRaML2-p140-C and p140-D.

Formulation Example 1

The following components were admixed in convention method and punched out to obtain 100 tablets each containing 5 mg of active ingredient.

Vitamin K₅ 500.0 mg Carboxymethylcellulose calcium 200.0 mg Magnesium stearate 100.0 mg Microcrystalline cellulose  9.2 mg

16 993 amino acids amino acid linear protein rat skeletal muscle myoblast L6 1 Met Glu Asn Pro Tyr Val Gly Arg Ala Arg Ala Ala Ala Glu Arg Ala 1 5 10 15 Ala Ala Glu Ala Thr Asn Ser Leu Ser Ile Leu Val Arg Pro Thr Ser 20 25 30 Glu Gly Ser Arg Ile Asp Ser Glu Phe Val Glu Leu Ala Trp Thr Ser 35 40 45 His Pro Glu Ser Gly Trp Glu Glu Val Ser Ala Tyr Asp Glu Ala Met 50 55 60 Asn Pro Ile Arg Thr Tyr Gln Val Cys Asn Val Arg Glu Ser Ser Gln 65 70 75 80 Asn Asn Trp Leu Arg Thr Gly Phe Ile Trp Arg Arg Glu Val Gln Arg 85 90 95 Val Tyr Val Glu Leu Lys Phe Thr Val Arg Asp Cys Asn Ser Ile Pro 100 105 110 Asn Ile Pro Gly Ser Cys Lys Glu Thr Phe Asn Leu Phe Tyr Tyr Glu 115 120 125 Ala Asp Ser Asp Val Ala Ser Ala Ser Ser Pro Phe Trp Met Glu Asn 130 135 140 Pro Tyr Val Lys Val Asp Thr Ile Ala Pro Asp Glu Ser Phe Ser Arg 145 150 155 160 Leu Asp Ala Gly Arg Val Asn Thr Lys Val Arg Ser Phe Gly Pro Leu 165 170 175 Ser Lys Ala Gly Phe Tyr Leu Ala Phe Gln Asp Gln Gly Ala Cys Met 180 185 190 Ser Leu Ile Ser Val Arg Ala Phe Tyr Lys Lys Cys Ala Ser Thr Thr 195 200 205 Ala Gly Phe Ala Leu Phe Pro Glu Thr Leu Thr Gly Ala Glu Pro Thr 210 215 220 Ser Leu Val Ile Ala Pro Gly Thr Cys Ile Ala Asn Ala Val Glu Val 225 230 235 240 Ser Val Pro Leu Lys Leu Tyr Cys Asn Gly Asp Gly Glu Trp Met Val 245 250 255 Pro Val Gly Ala Cys Thr Cys Ala Thr Gly His Glu Pro Ala Ala Lys 260 265 270 Glu Thr Gln Cys Arg Ala Cys Pro Pro Gly Ser Tyr Lys Ala Lys Gln 275 280 285 Gly Glu Gly Pro Cys Leu Pro Cys Pro Pro Asn Ser Arg Thr Thr Ser 290 295 300 Pro Ala Ala Ser Ile Cys Thr Cys His Asn Asn Phe Tyr Arg Ala Asp 305 310 315 320 Ser Asp Thr Ala Asp Ser Ala Cys Thr Thr Val Pro Ser Pro Pro Arg 325 330 335 Gly Val Ile Ser Asn Val Asn Glu Thr Ser Leu Ile Leu Glu Trp Ser 340 345 350 Glu Pro Arg Asp Leu Gly Gly Arg Asp Asp Leu Leu Tyr Asn Val Ile 355 360 365 Cys Lys Lys Cys Arg Gly Ser Ser Gly Ala Gly Gly Pro Ala Thr Cys 370 375 380 Ser Arg Cys Asp Asp Asn Val Glu Phe Glu Pro Arg Gln Leu Gly Leu 385 390 395 400 Thr Glu Arg Arg Val His Ile Ser His Leu Leu Ala His Thr Arg Tyr 405 410 415 Thr Phe Glu Val Gln Ala Val Asn Gly Val Ser Gly Lys Ser Pro Leu 420 425 430 Pro Pro Arg Tyr Ala Ala Val Asn Ile Thr Thr Asn Gln Ala Ala Pro 435 440 445 Ser Glu Val Pro Thr Leu His Leu His Ser Ser Ser Gly Ser Ser Leu 450 455 460 Thr Leu Ser Trp Ala Pro Pro Glu Arg Pro Asn Gly Val Ile Leu Asp 465 470 475 480 Tyr Glu Met Lys Tyr Phe Glu Lys Ser Lys Gly Ile Ala Ser Thr Val 485 490 495 Thr Ser Gln Lys Asn Ser Val Gln Leu Asp Gly Leu Gln Pro Asp Ala 500 505 510 Arg Tyr Val Val Gln Val Arg Ala Arg Thr Val Ala Gly Tyr Gly Gln 515 520 525 Tyr Ser Arg Pro Ala Glu Phe Glu Thr Thr Ser Glu Arg Gly Ser Gly 530 535 540 Ala Gln Gln Leu Gln Glu Gln Leu Pro Leu Ile Val Gly Ser Thr Val 545 550 555 560 Ala Gly Phe Val Phe Met Val Val Val Val Val Ile Ala Leu Val Cys 565 570 575 Leu Arg Lys Gln Arg Gln Gly Pro Asp Ala Glu Tyr Thr Glu Lys Leu 580 585 590 Gln Gln Tyr Val Ala Pro Arg Met Lys Val Tyr Ile Asp Pro Phe Thr 595 600 605 Tyr Glu Asp Pro Asn Glu Ala Val Arg Glu Phe Ala Lys Glu Ile Asp 610 615 620 Val Ser Cys Val Lys Ile Glu Glu Val Ile Gly Ala Gly Glu Phe Gly 625 630 635 640 Glu Val Cys Arg Gly Arg Leu Lys Leu Pro Gly Arg Arg Glu Val Phe 645 650 655 Val Ala Ile Lys Thr Leu Lys Val Gly Tyr Thr Glu Arg Gln Arg Arg 660 665 670 Asp Phe Leu Ser Glu Ala Ser Ile Met Gly Gln Phe Asp His Pro Asn 675 680 685 Ile Ile Arg Leu Glu Gly Val Val Thr Lys Ser Arg Pro Val Met Ile 690 695 700 Leu Thr Glu Phe Met Glu Asn Cys Ala Leu Asp Ser Phe Leu Arg Leu 705 710 715 720 Asn Asp Gly Gln Phe Thr Val Ile Gln Leu Val Gly Met Leu Arg Gly 725 730 735 Ile Ala Ala Gly Met Lys Tyr Leu Ser Glu Met Asn Tyr Val His Arg 740 745 750 Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys 755 760 765 Val Ser Asp Phe Gly Leu Ser Arg Phe Leu Glu Asp Asp Pro Ser Asp 770 775 780 Pro Thr Tyr Thr Ser Ser Leu Gly Gly Lys Ile Pro Ile Arg Trp Thr 785 790 795 800 Ala Pro Glu Ala Ile Asp Tyr Arg Lys Phe Thr Ser Ala Ser Asp Val 805 810 815 Trp Ser Tyr Gly Ile Val Met Trp Glu Val Met Ser Tyr Gly Glu Arg 820 825 830 Pro Tyr Trp Asp Met Ser Asn Gln Asp Val Ile Asn Ala Val Glu Gln 835 840 845 Asp Tyr Arg Leu Pro Pro Pro Met Asp Cys Pro Ala Ala Leu His Gln 850 855 860 Leu Met Leu Asp Cys Trp Val Arg Asp Arg Asn Leu Arg Pro Lys Phe 865 870 875 880 Ser Gln Ile Val Asn Thr Leu Asp Lys Leu Ile Arg Asn Ala Ala Ser 885 890 895 Leu Lys Val Ile Ala Ser Ala Pro Ser Gly Met Ser Gln Pro Leu Leu 900 905 910 Asp Arg Thr Val Pro Asp Tyr Thr Thr Phe Thr Thr Val Gly Asp Trp 915 920 925 Leu Asp Ala Ile Lys Met Gly Arg Tyr Lys Glu Ser Phe Val Gly Ala 930 935 940 Gly Phe Ala Ser Phe Asp Leu Val Ala Gln Met Thr Ala Glu Asp Leu 945 950 955 960 Leu Arg Ile Gly Val Thr Leu Ala Gly His Gln Lys Lys Ile Leu Ser 965 970 975 Ser Ile Gln Asp Met Arg Leu Gln Met Asn Gln Thr Leu Pro Val Gln 980 985 990 Val 2982 base pairs nucleic acid single linear cDNA to mRNA rat skeletal muscle myoblast L6 2 ATGGAGAACC CCTACGTTGG GCGAGCGAGA GCAGCAGCGG AGCGAGCAGC GGCAGAAGCC 60 ACGAATTCAC TATCGATCCT GGTTCGGCCC ACCTCTGAAG GTTCCAGAAT CGATAGTGAA 120 TTCGTGGAGC TGGCATGGAC ATCTCATCCA GAGAGTGGGT GGGAAGAAGT GAGCGCCTAC 180 GATGAAGCCA TGAATCCTAT CCGCACGTAT CAGGTGTGTA ACGTGCGCGA GTCCAGCCAG 240 AACAACTGGC TGCGGACCGG TTTCATCTGG CGGCGGGAAG TCCAGCGCGT CTACGTGGAG 300 CTGAAGTTTA CCGTGAGAGA TTGCAACAGC ATCCCCAACA TCCCTGGCTC CTGCAAGGAA 360 ACCTTCAACC TTTTTTACTA CGAGGCTGAT AGCGATGTGG CGTCAGCCTC CTCTCCCTTC 420 TGGATGGAGA ACCCCTACGT GAAAGTGGAC ACCATTGCGC CAGATGAGAG CTTCTCGCGG 480 CTAGACGCTG GGCGCGTTAA CACCAAAGTG CGCAGCTTCG GGCCGCTTTC CAAAGCCGGC 540 TTCTACTTGG CCTTCCAGGA CCAGGGTGCC TGCATGTCAC TCATCTCTGT GCGCGCCTTC 600 TACAAGAAGT GTGCATCCAC CACTGCAGGC TTCGCACTCT TCCCCGAGAC CCTCACGGGG 660 GCTGAGCCCA CTTCGCTGGT CATTGCCCCT GGCACCTGCA TCGCTAACGC TGTGGAGGTG 720 TCTGTACCGC TCAAGCTCTA CTGCAATGGC GACGGGGAGT GGATGGTGCC CGTTGGTGCC 780 TGCACCTGCG CTACTGGCCA TGAGCCAGCC GCCAAGGAGA CCCAGTGCCG CGCCTGTCCC 840 CCTGGGAGCT ACAAGGCAAA GCAAGGAGAG GGGCCCTGCC TCCCCTGTCC CCCCAATAGC 900 CGCACCACCT CGCCGGCTGC CAGCATCTGC ACCTGTCACA ATAATTTCTA CCGCGCAGAC 960 TCAGACACAG CGGACAGCGC CTGCACCACG GTGCCGTCTC CCCCCCGGGG TGTGATCTCC 1020 AATGTGAATG AGACCTCGCT GATCCTCGAG TGGAGTGAGC CCCGGGACCT TGGCGGACGA 1080 GATGACCTCC TTTATAATGT TATCTGTAAG AAGTGCCGTG GCAGCTCTGG GGCTGGAGGT 1140 CCGGCGACCT GTTCACGCTG TGATGACAAC GTGGAGTTCG AGCCCCGACA GCTGGGCCTG 1200 ACCGAGCGCC GGGTCCACAT CAGCCACCTG TTGGCCCACA CCCGCTACAC CTTTGAGGTG 1260 CAGGCTGTCA ACGGCGTCTC TGGCAAAAGC CCTTTGCCGC CCCGCTATGC AGCTGTGAAT 1320 ATCACCACCA ACCAGGCCGC CCCATCAGAA GTGCCTACGC TCCACTTGCA CAGCAGTTCA 1380 GGGAGCAGCC TGACCCTGTC CTGGGCACCC CCGGAGCGGC CTAACGGAGT CATCTTGGAC 1440 TATGAGATGA AGTACTTTGA GAAGAGTAAA GGCATCGCCT CCACTGTCAC CAGCCAGAAG 1500 AACTCTGTAC AACTGGACGG ACTGCAGCCC GACGCCCGCT ATGTAGTTCA GGTCCGGGCT 1560 CGCACAGTAG CAGGTTACGG ACAGTATAGC CGCCCAGCTG AGTTTGAGAC CACGAGTGAA 1620 AGAGGCTCAG GGGCCCAGCA GCTTCAAGAG CAGCTTCCCC TAATTGTGGG ATCCACCGTA 1680 GCTGGCTTTG TCTTCATGGT GGTCGTCGTG GTCATTGCTC TTGTCTGCCT CAGGAAGCAG 1740 CGCCAGGGCC CTGATGCAGA ATACACGGAG AAGTTGCAGC AATACGTTGC CCCCAGGATG 1800 AAAGTTTACA TTGACCCCTT TACCTACGAG GATCCCAATG AGGCCGTCCG AGAGTTCGCC 1860 AAGGAGATCG ATGTGTCCTG CGTCAAGATC GAGGAGGTGA TTGGAGCTGG GGAGTTTGGG 1920 GAAGTGTGCC GGGGTCGGCT GAAACTGCCC GGCCGCCGGG AGGTGTTCGT GGCCATCAAG 1980 ACACTGAAGG TGGGATACAC GGAGAGGCAG CGGCGGGACT TCCTGAGTGA GGCTTCCATC 2040 ATGGGTCAAT TTGACCATCC AAATATAATC CGTCTAGAGG GCGTGGTCAC CAAAAGTCGT 2100 CCAGTCATGA TCCTCACTGA GTTCATGGAG AACTGTGCCC TGGACTCCTT CCTACGGCTC 2160 AATGACGGGC AGTTCACAGT CATCCAGCTT GTGGGCATGT TGCGTGGCAT TGCTGCCGGC 2220 ATGAAGTACT TGTCTGAGAT GAACTACGTG CACCGTGACC TCGCTGCCCG CAACATCCTT 2280 GTCAACAGTA ACTTGGTCTG CAAAGTATCT GACTTTGGGC TCTCCCGCTT CCTGGAGGAC 2340 GACCCCTCAG ACCCCACCTA CACCAGCTCC CTGGGTGGGA AGATCCCTAT CCGTTGGACC 2400 GCCCCAGAGG CCATAGACTA TCGGAAGTTC ACGTCTGCCA GCGATGTCTG GAGCTACGGG 2460 ATCGTCATGT GGGAGGTCAT GAGCTACGGA GAGCGACCAT ACTGGGACAT GAGCAACCAG 2520 GATGTCATCA ATGCCGTAGA GCAAGACTAT CGGTTACCAC CCCCCATGGA CTGCCCAGCG 2580 GCGCTGCACC AGCTCATGCT GGACTGTTGG GTGCGGGACC GGAACCTCAG GCCCAAGTTC 2640 TCCCAAATCG TCAACACGCT AGACAAGCTT ATCCGCAATG CTGCCAGCCT CAAGGTCATC 2700 GCCAGTGCCC CATCTGGCAT GTCCCAGCCC CTCCTAGACC GCACGGTCCC AGATTATACG 2760 ACCTTCACGA CGGTGGGCGA CTGGCTAGAT GCCATCAAGA TGGGGAGGTA TAAAGAGAGC 2820 TTCGTCGGTG CGGGTTTTGC CTCCTTTGAC CTGGTGGCCC AGATGACTGC AGAAGATCTG 2880 CTAAGGATCG GGGTCACTTT GGCCGGCCAC CAGAAGAAGA TCCTCAGCAG TATCCAGGAC 2940 ATGCGGCTGC AGATGAACCA GACACTGCCC GTGCAGGTCT GA 2982 4027 base pairs nucleic acid single linear cDNA to mRNA rat skeletal muscle myoblast L6 3 GAAAAATGAA GATCTATACC GACAGCAGAT CAGTGGCTGC CTGGGGCAAA GTTGGAGGGA 60 CATGTTATTT TGATTGTGAT GACATAATAC ATGCAAACAC GGCTAATCCT CTCAAAGCAT 120 ACACTTATAC ATGTGCAGCT TGGTATACAT AAATTATCCA TTACAAAACT ATGAGAAAGC 180 TATCACCACT ATGAAGCACC ACTCACAGTA TGTGAATCTC CACCCCCCTT CCACTGCTGA 240 GACACAGAAA TCCTAGACTG GATGGAGAAC CCCTACGTTG GGCGAGCGAG AGCAGCAGCG 300 GAGCGAGCAG CGGCAGAAGC CACGAATTCA CTATCGATCC TGGTTCGGCC CACCTCTGAA 360 GGTTCCAGAA TCGATAGTGA ATTCGTGGAG CTGGCATGGA CATCTCATCC AGAGAGTGGG 420 TGGGAAGAAG TGAGCGCCTA CGATGAAGCC ATGAATCCTA TCCGCACGTA TCAGGTGTGT 480 AACGTGCGCG AGTCCAGCCA GAACAACTGG CTGCGGACCG GTTTCATCTG GCGGCGGGAA 540 GTCCAGCGCG TCTACGTGGA GCTGAAGTTT ACCGTGAGAG ATTGCAACAG CATCCCCAAC 600 ATCCCTGGCT CCTGCAAGGA AACCTTCAAC CTTTTTTACT ACGAGGCTGA TAGCGATGTG 660 GCGTCAGCCT CCTCTCCCTT CTGGATGGAG AACCCCTACG TGAAAGTGGA CACCATTGCG 720 CCAGATGAGA GCTTCTCGCG GCTAGACGCT GGGCGCGTTA ACACCAAAGT GCGCAGCTTC 780 GGGCCGCTTT CCAAAGCCGG CTTCTACTTG GCCTTCCAGG ACCAGGGTGC CTGCATGTCA 840 CTCATCTCTG TGCGCGCCTT CTACAAGAAG TGTGCATCCA CCACTGCAGG CTTCGCACTC 900 TTCCCCGAGA CCCTCACGGG GGCTGAGCCC ACTTCGCTGG TCATTGCCCC TGGCACCTGC 960 ATCGCTAACG CTGTGGAGGT GTCTGTACCG CTCAAGCTCT ACTGCAATGG CGACGGGGAG 1020 TGGATGGTGC CCGTTGGTGC CTGCACCTGC GCTACTGGCC ATGAGCCAGC CGCCAAGGAG 1080 ACCCAGTGCC GCGCCTGTCC CCCTGGGAGC TACAAGGCAA AGCAAGGAGA GGGGCCCTGC 1140 CTCCCCTGTC CCCCCAATAG CCGCACCACC TCGCCGGCTG CCAGCATCTG CACCTGTCAC 1200 AATAATTTCT ACCGCGCAGA CTCAGACACA GCGGACAGCG CCTGCACCAC GGTGCCGTCT 1260 CCCCCCCGGG GTGTGATCTC CAATGTGAAT GAGACCTCGC TGATCCTCGA GTGGAGTGAG 1320 CCCCGGGACC TTGGCGGACG AGATGACCTC CTTTATAATG TTATCTGTAA GAAGTGCCGT 1380 GGCAGCTCTG GGGCTGGAGG TCCGGCGACC TGTTCACGCT GTGATGACAA CGTGGAGTTC 1440 GAGCCCCGAC AGCTGGGCCT GACCGAGCGC CGGGTCCACA TCAGCCACCT GTTGGCCCAC 1500 ACCCGCTACA CCTTTGAGGT GCAGGCTGTC AACGGCGTCT CTGGCAAAAG CCCTTTGCCG 1560 CCCCGCTATG CAGCTGTGAA TATCACCACC AACCAGGCCG CCCCATCAGA AGTGCCTACG 1620 CTCCACTTGC ACAGCAGTTC AGGGAGCAGC CTGACCCTGT CCTGGGCACC CCCGGAGCGG 1680 CCTAACGGAG TCATCTTGGA CTATGAGATG AAGTACTTTG AGAAGAGTAA AGGCATCGCC 1740 TCCACTGTCA CCAGCCAGAA GAACTCTGTA CAACTGGACG GACTGCAGCC CGACGCCCGC 1800 TATGTAGTTC AGGTCCGGGC TCGCACAGTA GCAGGTTACG GACAGTATAG CCGCCCAGCT 1860 GAGTTTGAGA CCACGAGTGA AAGAGGCTCA GGGGCCCAGC AGCTTCAAGA GCAGCTTCCC 1920 CTAATTGTGG GATCCACCGT AGCTGGCTTT GTCTTCATGG TGGTCGTCGT GGTCATTGCT 1980 CTTGTCTGCC TCAGGAAGCA GCGCCAGGGC CCTGATGCAG AATACACGGA GAAGTTGCAG 2040 CAATACGTTG CCCCCAGGAT GAAAGTTTAC ATTGACCCCT TTACCTACGA GGATCCCAAT 2100 GAGGCCGTCC GAGAGTTCGC CAAGGAGATC GATGTGTCCT GCGTCAAGAT CGAGGAGGTG 2160 ATTGGAGCTG GGGAGTTTGG GGAAGTGTGC CGGGGTCGGC TGAAACTGCC CGGCCGCCGG 2220 GAGGTGTTCG TGGCCATCAA GACACTGAAG GTGGGATACA CGGAGAGGCA GCGGCGGGAC 2280 TTCCTGAGTG AGGCTTCCAT CATGGGTCAA TTTGACCATC CAAATATAAT CCGTCTAGAG 2340 GGCGTGGTCA CCAAAAGTCG TCCAGTCATG ATCCTCACTG AGTTCATGGA GAACTGTGCC 2400 CTGGACTCCT TCCTACGGCT CAATGACGGG CAGTTCACAG TCATCCAGCT TGTGGGCATG 2460 TTGCGTGGCA TTGCTGCCGG CATGAAGTAC TTGTCTGAGA TGAACTACGT GCACCGTGAC 2520 CTCGCTGCCC GCAACATCCT TGTCAACAGT AACTTGGTCT GCAAAGTATC TGACTTTGGG 2580 CTCTCCCGCT TCCTGGAGGA CGACCCCTCA GACCCCACCT ACACCAGCTC CCTGGGTGGG 2640 AAGATCCCTA TCCGTTGGAC CGCCCCAGAG GCCATAGACT ATCGGAAGTT CACGTCTGCC 2700 AGCGATGTCT GGAGCTACGG GATCGTCATG TGGGAGGTCA TGAGCTACGG AGAGCGACCA 2760 TACTGGGACA TGAGCAACCA GGATGTCATC AATGCCGTAG AGCAAGACTA TCGGTTACCA 2820 CCCCCCATGG ACTGCCCAGC GGCGCTGCAC CAGCTCATGC TGGACTGTTG GGTGCGGGAC 2880 CGGAACCTCA GGCCCAAGTT CTCCCAAATC GTCAACACGC TAGACAAGCT TATCCGCAAT 2940 GCTGCCAGCC TCAAGGTCAT CGCCAGTGCC CCATCTGGCA TGTCCCAGCC CCTCCTAGAC 3000 CGCACGGTCC CAGATTATAC GACCTTCACG ACGGTGGGCG ACTGGCTAGA TGCCATCAAG 3060 ATGGGGAGGT ATAAAGAGAG CTTCGTCGGT GCGGGTTTTG CCTCCTTTGA CCTGGTGGCC 3120 CAGATGACTG CAGAAGATCT GCTAAGGATC GGGGTCACTT TGGCCGGCCA CCAGAAGAAG 3180 ATCCTCAGCA GTATCCAGGA CATGCGGCTG CAGATGAACC AGACACTGCC CGTGCAGGTC 3240 TGACGCTCAG CTCCAGCGAG GGGCGTGGCC CCCCGGGACT GCACAAGGAT TCTGACCAGC 3300 CAGCTGGACT TTTGGATACC TGGCCTTTGG CTGTGGCCCA GAAGACAGAA GTTCGGGGGA 3360 GAACCCTAGC TGTGACTTCT CCAAGCCTGT GCTCCCTCCC AGGAAGTGTG CCCCAAACCT 3420 CTTCATATTG AAGATGGATT AGAAGAGGGG GTGATATCCC CTCCCCAGAT GCCTCAGGGC 3480 CCAGGCCTGC CTGCTCTCCA GTCGGGGATC TTCACAACTC AGATTTGGTT GTGCTTCAGT 3540 AGTGGAGGTC CTGGTAGGGT CGGGTGGGGA TAAGCCTGGG TTCTTCAGGC CCCAGCCCTG 3600 GCAGGGGTCT GACCCCAGCA GGTAAGCAGA GAGTACTCCC TCCCCAGGAA GTGGAGGAGG 3660 GGACTCTGGG AATGGGGAAA TATGGTGCCC CATCCTGAAG CCAGCTGGTA CCTCCAGTTT 3720 GCACAGGGAC TTGTTGGGGG CTGAGGGCCC TGCCTACCCT TGGTGCTGTC ATAAAAGGGC 3780 AGGCGGGAGC GGGCTGAGAA ACAGCCTGTG CCTCCCAGAG ACTGACTCAG AGAGCCAGAG 3840 ACGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGTGT GTGTGTGAAA GACGGGGGTG 3900 GGGTATGTAT GCGTGTGTTG TGCACATGCT TGCCTGCACA GAGAGCATGA GTGTGTACAA 3960 GCTTAGCCCT GTGCCCTGTA GTGGGGCCAG CTGGGCAGAC AGCGAAATAA AAGGCAATAA 4020 GTTGAAA 4027 4027 base pairs nucleic acid single linear cDNA to mRNA rat skeletal muscle myoblast L6 CDS 262..3243 by similarity to some other pattern 4 GAAAAATGAA GATCTATACC GACAGCAGAT CAGTGGCTGC CTGGGGCAAA GTTGGAGGGA 60 CATGTTATTT TGATTGTGAT GACATAATAC ATGCAAACAC GGCTAATCCT CTCAAAGCAT 120 ACACTTATAC ATGTGCAGCT TGGTATACAT AAATTATCCA TTACAAAACT ATGAGAAAGC 180 TATCACCACT ATGAAGCACC ACTCACAGTA TGTGAATCTC CACCCCCCTT CCACTGCTGA 240 GACACAGAAA TCCTAGACTG G ATG GAG AAC CCC TAC GTT GGG CGA GCG AGA 291 Met Glu Asn Pro Tyr Val Gly Arg Ala Arg 1 5 10 GCA GCA GCG GAG CGA GCA GCG GCA GAA GCC ACG AAT TCA CTA TCG ATC 339 Ala Ala Ala Glu Arg Ala Ala Ala Glu Ala Thr Asn Ser Leu Ser Ile 15 20 25 CTG GTT CGG CCC ACC TCT GAA GGT TCC AGA ATC GAT AGT GAA TTC GTG 387 Leu Val Arg Pro Thr Ser Glu Gly Ser Arg Ile Asp Ser Glu Phe Val 30 35 40 GAG CTG GCA TGG ACA TCT CAT CCA GAG AGT GGG TGG GAA GAA GTG AGC 435 Glu Leu Ala Trp Thr Ser His Pro Glu Ser Gly Trp Glu Glu Val Ser 45 50 55 GCC TAC GAT GAA GCC ATG AAT CCT ATC CGC ACG TAT CAG GTG TGT AAC 483 Ala Tyr Asp Glu Ala Met Asn Pro Ile Arg Thr Tyr Gln Val Cys Asn 60 65 70 GTG CGC GAG TCC AGC CAG AAC AAC TGG CTG CGG ACC GGT TTC ATC TGG 531 Val Arg Glu Ser Ser Gln Asn Asn Trp Leu Arg Thr Gly Phe Ile Trp 75 80 85 90 CGG CGG GAA GTC CAG CGC GTC TAC GTG GAG CTG AAG TTT ACC GTG AGA 579 Arg Arg Glu Val Gln Arg Val Tyr Val Glu Leu Lys Phe Thr Val Arg 95 100 105 GAT TGC AAC AGC ATC CCC AAC ATC CCT GGC TCC TGC AAG GAA ACC TTC 627 Asp Cys Asn Ser Ile Pro Asn Ile Pro Gly Ser Cys Lys Glu Thr Phe 110 115 120 AAC CTT TTT TAC TAC GAG GCT GAT AGC GAT GTG GCG TCA GCC TCC TCT 675 Asn Leu Phe Tyr Tyr Glu Ala Asp Ser Asp Val Ala Ser Ala Ser Ser 125 130 135 CCC TTC TGG ATG GAG AAC CCC TAC GTG AAA GTG GAC ACC ATT GCG CCA 723 Pro Phe Trp Met Glu Asn Pro Tyr Val Lys Val Asp Thr Ile Ala Pro 140 145 150 GAT GAG AGC TTC TCG CGG CTA GAC GCT GGG CGC GTT AAC ACC AAA GTG 771 Asp Glu Ser Phe Ser Arg Leu Asp Ala Gly Arg Val Asn Thr Lys Val 155 160 165 170 CGC AGC TTC GGG CCG CTT TCC AAA GCC GGC TTC TAC TTG GCC TTC CAG 819 Arg Ser Phe Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu Ala Phe Gln 175 180 185 GAC CAG GGT GCC TGC ATG TCA CTC ATC TCT GTG CGC GCC TTC TAC AAG 867 Asp Gln Gly Ala Cys Met Ser Leu Ile Ser Val Arg Ala Phe Tyr Lys 190 195 200 AAG TGT GCA TCC ACC ACT GCA GGC TTC GCA CTC TTC CCC GAG ACC CTC 915 Lys Cys Ala Ser Thr Thr Ala Gly Phe Ala Leu Phe Pro Glu Thr Leu 205 210 215 ACG GGG GCT GAG CCC ACT TCG CTG GTC ATT GCC CCT GGC ACC TGC ATC 963 Thr Gly Ala Glu Pro Thr Ser Leu Val Ile Ala Pro Gly Thr Cys Ile 220 225 230 GCT AAC GCT GTG GAG GTG TCT GTA CCG CTC AAG CTC TAC TGC AAT GGC 1011 Ala Asn Ala Val Glu Val Ser Val Pro Leu Lys Leu Tyr Cys Asn Gly 235 240 245 250 GAC GGG GAG TGG ATG GTG CCC GTT GGT GCC TGC ACC TGC GCT ACT GGC 1059 Asp Gly Glu Trp Met Val Pro Val Gly Ala Cys Thr Cys Ala Thr Gly 255 260 265 CAT GAG CCA GCC GCC AAG GAG ACC CAG TGC CGC GCC TGT CCC CCT GGG 1107 His Glu Pro Ala Ala Lys Glu Thr Gln Cys Arg Ala Cys Pro Pro Gly 270 275 280 AGC TAC AAG GCA AAG CAA GGA GAG GGG CCC TGC CTC CCC TGT CCC CCC 1155 Ser Tyr Lys Ala Lys Gln Gly Glu Gly Pro Cys Leu Pro Cys Pro Pro 285 290 295 AAT AGC CGC ACC ACC TCG CCG GCT GCC AGC ATC TGC ACC TGT CAC AAT 1203 Asn Ser Arg Thr Thr Ser Pro Ala Ala Ser Ile Cys Thr Cys His Asn 300 305 310 AAT TTC TAC CGC GCA GAC TCA GAC ACA GCG GAC AGC GCC TGC ACC ACG 1251 Asn Phe Tyr Arg Ala Asp Ser Asp Thr Ala Asp Ser Ala Cys Thr Thr 315 320 325 330 GTG CCG TCT CCC CCC CGG GGT GTG ATC TCC AAT GTG AAT GAG ACC TCG 1299 Val Pro Ser Pro Pro Arg Gly Val Ile Ser Asn Val Asn Glu Thr Ser 335 340 345 CTG ATC CTC GAG TGG AGT GAG CCC CGG GAC CTT GGC GGA CGA GAT GAC 1347 Leu Ile Leu Glu Trp Ser Glu Pro Arg Asp Leu Gly Gly Arg Asp Asp 350 355 360 CTC CTT TAT AAT GTT ATC TGT AAG AAG TGC CGT GGC AGC TCT GGG GCT 1395 Leu Leu Tyr Asn Val Ile Cys Lys Lys Cys Arg Gly Ser Ser Gly Ala 365 370 375 GGA GGT CCG GCG ACC TGT TCA CGC TGT GAT GAC AAC GTG GAG TTC GAG 1443 Gly Gly Pro Ala Thr Cys Ser Arg Cys Asp Asp Asn Val Glu Phe Glu 380 385 390 CCC CGA CAG CTG GGC CTG ACC GAG CGC CGG GTC CAC ATC AGC CAC CTG 1491 Pro Arg Gln Leu Gly Leu Thr Glu Arg Arg Val His Ile Ser His Leu 395 400 405 410 TTG GCC CAC ACC CGC TAC ACC TTT GAG GTG CAG GCT GTC AAC GGC GTC 1539 Leu Ala His Thr Arg Tyr Thr Phe Glu Val Gln Ala Val Asn Gly Val 415 420 425 TCT GGC AAA AGC CCT TTG CCG CCC CGC TAT GCA GCT GTG AAT ATC ACC 1587 Ser Gly Lys Ser Pro Leu Pro Pro Arg Tyr Ala Ala Val Asn Ile Thr 430 435 440 ACC AAC CAG GCC GCC CCA TCA GAA GTG CCT ACG CTC CAC TTG CAC AGC 1635 Thr Asn Gln Ala Ala Pro Ser Glu Val Pro Thr Leu His Leu His Ser 445 450 455 AGT TCA GGG AGC AGC CTG ACC CTG TCC TGG GCA CCC CCG GAG CGG CCT 1683 Ser Ser Gly Ser Ser Leu Thr Leu Ser Trp Ala Pro Pro Glu Arg Pro 460 465 470 AAC GGA GTC ATC TTG GAC TAT GAG ATG AAG TAC TTT GAG AAG AGT AAA 1731 Asn Gly Val Ile Leu Asp Tyr Glu Met Lys Tyr Phe Glu Lys Ser Lys 475 480 485 490 GGC ATC GCC TCC ACT GTC ACC AGC CAG AAG AAC TCT GTA CAA CTG GAC 1779 Gly Ile Ala Ser Thr Val Thr Ser Gln Lys Asn Ser Val Gln Leu Asp 495 500 505 GGA CTG CAG CCC GAC GCC CGC TAT GTA GTT CAG GTC CGG GCT CGC ACA 1827 Gly Leu Gln Pro Asp Ala Arg Tyr Val Val Gln Val Arg Ala Arg Thr 510 515 520 GTA GCA GGT TAC GGA CAG TAT AGC CGC CCA GCT GAG TTT GAG ACC ACG 1875 Val Ala Gly Tyr Gly Gln Tyr Ser Arg Pro Ala Glu Phe Glu Thr Thr 525 530 535 AGT GAA AGA GGC TCA GGG GCC CAG CAG CTT CAA GAG CAG CTT CCC CTA 1923 Ser Glu Arg Gly Ser Gly Ala Gln Gln Leu Gln Glu Gln Leu Pro Leu 540 545 550 ATT GTG GGA TCC ACC GTA GCT GGC TTT GTC TTC ATG GTG GTC GTC GTG 1971 Ile Val Gly Ser Thr Val Ala Gly Phe Val Phe Met Val Val Val Val 555 560 565 570 GTC ATT GCT CTT GTC TGC CTC AGG AAG CAG CGC CAG GGC CCT GAT GCA 2019 Val Ile Ala Leu Val Cys Leu Arg Lys Gln Arg Gln Gly Pro Asp Ala 575 580 585 GAA TAC ACG GAG AAG TTG CAG CAA TAC GTT GCC CCC AGG ATG AAA GTT 2067 Glu Tyr Thr Glu Lys Leu Gln Gln Tyr Val Ala Pro Arg Met Lys Val 590 595 600 TAC ATT GAC CCC TTT ACC TAC GAG GAT CCC AAT GAG GCC GTC CGA GAG 2115 Tyr Ile Asp Pro Phe Thr Tyr Glu Asp Pro Asn Glu Ala Val Arg Glu 605 610 615 TTC GCC AAG GAG ATC GAT GTG TCC TGC GTC AAG ATC GAG GAG GTG ATT 2163 Phe Ala Lys Glu Ile Asp Val Ser Cys Val Lys Ile Glu Glu Val Ile 620 625 630 GGA GCT GGG GAG TTT GGG GAA GTG TGC CGG GGT CGG CTG AAA CTG CCC 2211 Gly Ala Gly Glu Phe Gly Glu Val Cys Arg Gly Arg Leu Lys Leu Pro 635 640 645 650 GGC CGC CGG GAG GTG TTC GTG GCC ATC AAG ACA CTG AAG GTG GGA TAC 2259 Gly Arg Arg Glu Val Phe Val Ala Ile Lys Thr Leu Lys Val Gly Tyr 655 660 665 ACG GAG AGG CAG CGG CGG GAC TTC CTG AGT GAG GCT TCC ATC ATG GGT 2307 Thr Glu Arg Gln Arg Arg Asp Phe Leu Ser Glu Ala Ser Ile Met Gly 670 675 680 CAA TTT GAC CAT CCA AAT ATA ATC CGT CTA GAG GGC GTG GTC ACC AAA 2355 Gln Phe Asp His Pro Asn Ile Ile Arg Leu Glu Gly Val Val Thr Lys 685 690 695 AGT CGT CCA GTC ATG ATC CTC ACT GAG TTC ATG GAG AAC TGT GCC CTG 2403 Ser Arg Pro Val Met Ile Leu Thr Glu Phe Met Glu Asn Cys Ala Leu 700 705 710 GAC TCC TTC CTA CGG CTC AAT GAC GGG CAG TTC ACA GTC ATC CAG CTT 2451 Asp Ser Phe Leu Arg Leu Asn Asp Gly Gln Phe Thr Val Ile Gln Leu 715 720 725 730 GTG GGC ATG TTG CGT GGC ATT GCT GCC GGC ATG AAG TAC TTG TCT GAG 2499 Val Gly Met Leu Arg Gly Ile Ala Ala Gly Met Lys Tyr Leu Ser Glu 735 740 745 ATG AAC TAC GTG CAC CGT GAC CTC GCT GCC CGC AAC ATC CTT GTC AAC 2547 Met Asn Tyr Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn 750 755 760 AGT AAC TTG GTC TGC AAA GTA TCT GAC TTT GGG CTC TCC CGC TTC CTG 2595 Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Phe Leu 765 770 775 GAG GAC GAC CCC TCA GAC CCC ACC TAC ACC AGC TCC CTG GGT GGG AAG 2643 Glu Asp Asp Pro Ser Asp Pro Thr Tyr Thr Ser Ser Leu Gly Gly Lys 780 785 790 ATC CCT ATC CGT TGG ACC GCC CCA GAG GCC ATA GAC TAT CGG AAG TTC 2691 Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Asp Tyr Arg Lys Phe 795 800 805 810 ACG TCT GCC AGC GAT GTC TGG AGC TAC GGG ATC GTC ATG TGG GAG GTC 2739 Thr Ser Ala Ser Asp Val Trp Ser Tyr Gly Ile Val Met Trp Glu Val 815 820 825 ATG AGC TAC GGA GAG CGA CCA TAC TGG GAC ATG AGC AAC CAG GAT GTC 2787 Met Ser Tyr Gly Glu Arg Pro Tyr Trp Asp Met Ser Asn Gln Asp Val 830 835 840 ATC AAT GCC GTA GAG CAA GAC TAT CGG TTA CCA CCC CCC ATG GAC TGC 2835 Ile Asn Ala Val Glu Gln Asp Tyr Arg Leu Pro Pro Pro Met Asp Cys 845 850 855 CCA GCG GCG CTG CAC CAG CTC ATG CTG GAC TGT TGG GTG CGG GAC CGG 2883 Pro Ala Ala Leu His Gln Leu Met Leu Asp Cys Trp Val Arg Asp Arg 860 865 870 AAC CTC AGG CCC AAG TTC TCC CAA ATC GTC AAC ACG CTA GAC AAG CTT 2931 Asn Leu Arg Pro Lys Phe Ser Gln Ile Val Asn Thr Leu Asp Lys Leu 875 880 885 890 ATC CGC AAT GCT GCC AGC CTC AAG GTC ATC GCC AGT GCC CCA TCT GGC 2979 Ile Arg Asn Ala Ala Ser Leu Lys Val Ile Ala Ser Ala Pro Ser Gly 895 900 905 ATG TCC CAG CCC CTC CTA GAC CGC ACG GTC CCA GAT TAT ACG ACC TTC 3027 Met Ser Gln Pro Leu Leu Asp Arg Thr Val Pro Asp Tyr Thr Thr Phe 910 915 920 ACG ACG GTG GGC GAC TGG CTA GAT GCC ATC AAG ATG GGG AGG TAT AAA 3075 Thr Thr Val Gly Asp Trp Leu Asp Ala Ile Lys Met Gly Arg Tyr Lys 925 930 935 GAG AGC TTC GTC GGT GCG GGT TTT GCC TCC TTT GAC CTG GTG GCC CAG 3123 Glu Ser Phe Val Gly Ala Gly Phe Ala Ser Phe Asp Leu Val Ala Gln 940 945 950 ATG ACT GCA GAA GAT CTG CTA AGG ATC GGG GTC ACT TTG GCC GGC CAC 3171 Met Thr Ala Glu Asp Leu Leu Arg Ile Gly Val Thr Leu Ala Gly His 955 960 965 970 CAG AAG AAG ATC CTC AGC AGT ATC CAG GAC ATG CGG CTG CAG ATG AAC 3219 Gln Lys Lys Ile Leu Ser Ser Ile Gln Asp Met Arg Leu Gln Met Asn 975 980 985 CAG ACA CTG CCC GTG CAG GTC TGACGCTCAG CTCCAGCGAG GGGCGTGGCC 3270 Gln Thr Leu Pro Val Gln Val 990 CCCCGGGACT GCACAAGGAT TCTGACCAGC CAGCTGGACT TTTGGATACC TGGCCTTTGG 3330 CTGTGGCCCA GAAGACAGAA GTTCGGGGGA GAACCCTAGC TGTGACTTCT CCAAGCCTGT 3390 GCTCCCTCCC AGGAAGTGTG CCCCAAACCT CTTCATATTG AAGATGGATT AGAAGAGGGG 3450 GTGATATCCC CTCCCCAGAT GCCTCAGGGC CCAGGCCTGC CTGCTCTCCA GTCGGGGATC 3510 TTCACAACTC AGATTTGGTT GTGCTTCAGT AGTGGAGGTC CTGGTAGGGT CGGGTGGGGA 3570 TAAGCCTGGG TTCTTCAGGC CCCAGCCCTG GCAGGGGTCT GACCCCAGCA GGTAAGCAGA 3630 GAGTACTCCC TCCCCAGGAA GTGGAGGAGG GGACTCTGGG AATGGGGAAA TATGGTGCCC 3690 CATCCTGAAG CCAGCTGGTA CCTCCAGTTT GCACAGGGAC TTGTTGGGGG CTGAGGGCCC 3750 TGCCTACCCT TGGTGCTGTC ATAAAAGGGC AGGCGGGAGC GGGCTGAGAA ACAGCCTGTG 3810 CCTCCCAGAG ACTGACTCAG AGAGCCAGAG ACGTGTGTGT GTGTGTGTGT GTGTGTGTGT 3870 GTGTGTGTGT GTGTGTGAAA GACGGGGGTG GGGTATGTAT GCGTGTGTTG TGCACATGCT 3930 TGCCTGCACA GAGAGCATGA GTGTGTACAA GCTTAGCCCT GTGCCCTGTA GTGGGGCCAG 3990 CTGGGCAGAC AGCGAAATAA AAGGCAATAA GTTGAAA 4027 11 amino acids amino acid linear protein rat skeletal muscle myoblast L6 5 Val Ile Gly Ala Gly Glu Phe Gly Glu Val Cys 1 5 10 10 amino acids amino acid linear protein rat skeletal muscle myoblast L6 6 Asn Ile Leu Val Asn Ser Asn Leu Val Cys 1 5 10 5 amino acids amino acid linear protein rat skeletal muscle myoblast L6 7 Val Glu Gln Asp Tyr 1 5 28 base pairs nucleic acid single linear DNA (synthetic) not provided 8 GTAATACGAC TCACTATAGG GGAGAGCT 28 28 base pairs nucleic acid single linear DNA (synthetic) not provided 9 CTCCCCTATA GTGAGTCGTA TTACTGCA 28 32 base pairs nucleic acid single linear DNA (synthetic) not provided 10 CTAGTCTATA GTGTCACCTA AATCGTGGGT AC 32 23 base pairs nucleic acid single linear DNA (synthetic) not provided 11 CACGATTTAG GTGACACTAT AGA 23 44 base pairs nucleic acid single linear DNA (synthetic) not provided 12 AATATAGTCG ACCACCATGG AGAACCCCTA CGTTGGGCGA GCGA 44 37 base pairs nucleic acid single linear DNA (synthetic) not provided 13 CGGCGGACTA GTTCAGACCT GCACGGGCAG TGTCTGG 37 55 base pairs nucleic acid single linear DNA (synthetic) not provided 14 GCCGCCACTA GTTCAGTGGT GGTGGTGGTG GTGGACCTGC ACGGGCAGTG TCTGG 55 44 base pairs nucleic acid single linear DNA (synthetic) not provided 15 CGGCGGACTA GTTCATGAGC CTCTTTCACT CGTGGTCTCA AACT 44 62 base pairs nucleic acid single linear DNA (synthetic) not provided 16 GCCGCCACTA GTTCAGTGGT GGTGGTGGTG GTGTGAGCCT CTTTCACTCG TGGTCTCAAA 60 CT 62 

What is claimed is:
 1. A method of screening for an agent affecting glucose uptake, comprising the steps of: (a) administering an amount of an agent to be tested to cells in vitro; (b) culturing said cells under conditions such that relative to a control, an altered level of phosphorylated protein p140 consisting of the amino acid sequence set forth in SEQ ID NO: 1 is detected; and (c) wherein the screening of the agent is completed when said altered level of phosphorylated protein p140 is correlated with glucose uptake. 